Etanercept formulations exhibiting marked reduction in sub-visible particles

ABSTRACT

The invention provides stabilized aqueous pharmaceutical etanercept compositions suitable for long-term storage of etanercept, with substantial reduction in sub-visible particles, and methods of manufacture of these compositions, methods of administration, and articles of manufacture.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 14/592,569 filed onJan. 8, 2015, which is a continuation of 35 U.S.C. 371 National PhaseEntry Application PCT/US2013/49778, filed on Jul. 9, 2013, which claimsthe benefit of U.S. Ser. No. 61/806,235, filed on Mar. 28, 2013, andU.S. Ser. No. 61/669,480 filed on Jul. 9, 2012, the disclosures of whichare incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

The present invention relates to aqueous pharmaceutical compositionsstabilized for long-term storage of etanercept, methods of manufactureof the compositions, methods of their administration, and kitscontaining the same, wherein the stabilized etanercept compositionsexhibit significant reduction in sub-visible particles. The inventionincludes etanercept formulations that do not require arginine forstabilization. The invention is further directed to articles and methodsaffording reduced patient exposure to sub-visible particles inetanercept formulations.

BACKGROUND OF THE INVENTION

Polypeptides must often be stored prior to their use. When stored forextended periods, polypeptides are frequently unstable in solution(Manning et al., 1989, Pharm. Res. 6:903-918). To extend their shelflife, additional processing steps have been developed, such as drying,e.g., lyophilization. However, lyophilized pharmaceutical compositionsare less convenient to use.

Typical practices to improve polypeptide stability can be addressed byvarying the concentration of elements with the formulation, or by addingexcipients to modify the formulation (See, for example, U.S. Pat. Nos.5,580,856 and 6,171,586). However, the use of additives can still resultin inactive polypeptides. In addition, in the case of lyophilization,the rehydration step can result in inactivation of the polypeptide by,for example, aggregation or denaturation (Flora et al., 1992, Pharm.Res., 9:33-36; Liu et al., 1991, Biotechnol. Bioeng., 37:177-184).Aggregation of polypeptides is undesirable, as it may result inimmunogenicity (Cleland et al., 1993, Crit. Rev. Therapeutic DrugCarrier Systems, 10:307-377; and Robbins et al., 1987, Diabetes,36:838-845).

Another way to improve polypeptide stability is to use L-arginine at aspecific concentration (U.S. Pat. No. 7,648,702).

One of the polypeptides that is stored for up to two years prior to useis etanercept (Enbrel®, Immunex Corporation), which is a dimeric fusionpolypeptide consisting of the extracellular ligand-binding portion ofthe human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR)linked to the Fc portion of human IgG1. It consists of 934 amino acidsand has an apparent molecular weight of approximately 150 kilodaltons(Physicians Desk Reference, 2002, Medical Economics Company Inc.) The Fccomponent of etanercept contains the constant heavy 2 (CH2) domain, theconstant heavy 3 (CH3) domain and hinge region, but not the constantheavy 1 (CH1) domain of human IgG1. An Fc domain can contain one or allof the domains described above. Etanercept is usually produced byrecombinant DNA technology in a Chinese hamster ovary (CHO) mammaliancell expression system.

A known problem in pharmaceutical protein formulations, includingformulations of etanercept, is the tendency for such formulations toexhibit the presence of sub-visible particles. Such particles arebelieved to be associated with undesired immune reactions(immunogenicity) when protein formulations are administered to patients.Such immune reactions can reduce the effectiveness of the proteintherapeutic, and may be harmful to the patient.

The present invention provides novel stable liquid formulations ofetanercept that allow its long-term storage and a marked reduction inthe tendency to form sub-visible particles.

SUMMARY OF THE INVENTION

The present invention provides an aqueous pharmaceutical compositioncomprising etanercept and a stabilizer for preventing, inhibiting orreducing the occurrence of subvisible particles in the composition,wherein the stabilizer comprises a surfactant, preferably selected fromthe group consisting of polysorbate surfactants and poloxamersurfactants, wherein said surfactant is capable of affording asignificant reduction in sub-visible particles in the composition incomparison to an etanercept composition lacking such surfactant. Theinvention contemplates the use of any pharmaceutically acceptablesurfactant capable of achieving the desired reduction in sub-visibleparticles.

In a further embodiment, the present invention is an aqueouspharmaceutical composition comprising etanercept and a stabilizer forpreventing, inhibiting or reducing the occurrence of sub-visibleparticles in the composition, wherein the stabilizer comprises asurfactant selected from the group consisting of polysorbate surfactantsand poloxamer surfactants. The stabilizer comprises a polysorbatesurfactant selected from the group consisting of polysorbate 80,polysorbate 60, polysorbate 40 and polysorbate 20, or a poloxamersurfactant, such as for example Pluronic F-68. Surprisingly, theformulations of the present invention exhibit excellent stability andreduction in sub-visible particles without need for the argininestabilizer present in commercial Enbrel® formulations. Accordingly, theformulations preferably contain no arginine, or are essentially free ofarginine.

In further embodiments of the invention, the etanercept formulationscontaining a polysorbate or poloxamer surfactant for stabilizationagainst sub-visible particle formation may further comprise anadditional stabilizing ingredient selected from: (i) an amino acidselected from the group consisting of serine, proline and glutamate;(ii) a metal ion selected from the group consisting calcium, magnesiumand zinc; (iii) a xylitol stabilizer selected from xylitol, alone, or acombination of xylitol and meglumine; (iv) a sodium chloride stabilizerselected from sodium chloride alone; sodium chloride in combination withsucrose or trehalose, and the combination of sodium chloride, sucroseand trehalose; (v) a meglumine stabilizer selected from meglumine alone;meglumine in combination with sucrose; meglumine in combination withsodium chloride; and meglumine with sodium chloride and sucrose; and(vi) the combination of a sugar and a polyol.

Thus in one embodiment, the invention provides a stable aqueouspharmaceutical composition comprising etanercept, a polysorbate orpoloxamer surfactant for stabilization against sub-visible particleformation, and a further stabilizer ingredient to inhibit instability,aggregation and/or fragmentation of the etanercept, wherein the furtherstabilizer comprises a compound selected from the group consisting ofserine, proline and glutamate. In a preferred embodiment, the stabilizercomprises glutamate.

In another embodiment, the invention provides a stable aqueouspharmaceutical composition comprising etanercept, a polysorbate orpoloxamer surfactant for stabilization against sub-visible particleformation, and a further stabilizer to inhibit instability, aggregationand/or fragmentation of the etanercept, wherein said further stabilizercomprises a stabilizing metal ion. Preferably, the surfactant isPolysorbate 80 or Pluronic F-68, and the metal ion is selected from thegroup consisting of calcium, magnesium, zinc, and combinations thereof.In an even more preferred embodiment, calcium, magnesium, zinc areprovided as calcium chloride, magnesium chloride and zinc chloride,respectively. Calcium chloride and magnesium chloride are particularlypreferred as stabilizers for etanercept.

In a further embodiment, the invention provides a stable aqueouspharmaceutical composition comprising etanercept, a polysorbate orpoloxamer surfactant for stabilization against sub-visible particleformation, and a further stabilizer ingredient to inhibit instability,aggregation and/or fragmentation of the etanercept, wherein said furtherstabilizer is selected from the group consisting of ionic polyolderivatives, such as meglumine, mannosylglycerate, glucosylglycerate,mannosyllactate, mannosylglycolate, and diglycerolphosphate. In thisembodiment, a preferred aqueous stabilized formulation of etanerceptcomprises: etanercept; polysorbate 80 surfactant or Pluronic F-68surfactant; and stabilizing ingredients to retard instability,aggregation and fragmentation of the etanercept in the formulation, saidstabilizing ingredients being comprised of (a) meglumine; or (b)meglumine in combination with sucrose; or (c) meglumine in combinationwith sodium chloride; or (d) meglumine in combination with sodiumchloride and sucrose.

In yet another embodiment, the invention provides a stable aqueouspharmaceutical composition comprising etanercept, a polysorbate orpoloxamer surfactant for stabilization against sub-visible particleformation, and a further stabilizer ingredient to inhibit instability,aggregation and/or fragmentation of the etanercept, wherein said furtherstabilizer comprises the combination of a sugar and a polyol.Preferably, surfactant is PS-80 or Pluronic F-68, the sugar is sucroseand the polyol is selected from the group consisting of mannitol andsorbitol. In a further aspect of this embodiment, the sugar is dextroseand the polyol is selected from the group consisting of mannitol andsorbitol. In a particularly preferred example of this embodiment, theinvention is directed to stabilized etanercept formulation wherein acombination of sucrose and mannitol is present to provide stabilizationof the etanercept monomer.

In a further embodiment, the invention provides a stable aqueouspharmaceutical composition comprising etanercept, a polysorbate orpoloxamer surfactant for stabilization against sub-visible particleformation, and wherein the invention provides a further ingredient forstabilization to reduce instability, aggregation and/or fragmentation ofthe etanercept, said formulation comprising about 25 to about 75 mg/mlof etanercept and one or more stabilizers, wherein the furtherstabilizer ingredients are selected from the group consisting of (i)sodium chloride and (ii) sodium chloride in combination with sucrose ortrehalose; and (iii) a combination of sodium chloride, sucrose andtrehalose; and wherein the stabilizer/surfactant for reduction insub-visible particles is preferably PS 80 or Pluronic F-68.

In yet another embodiment, the invention provides a stable aqueouspharmaceutical composition comprising etanercept, a polysorbate orpoloxamer surfactant, preferably PS-80 or Pluronic F-68, forstabilization against sub-visible particle formation, and where theinvention provides a further stabilizing ingredient to inhibitinstability, aggregation and/or fragmentation of the etanercept, whereinthe further stabilizer comprises xylitol or a combination of xylitol andmeglumine.

The invention is further directed to a method for reducing the number ofsub-visible particles in an etanercept formulation, said methodcomprising the step of adding a surfactant, preferably a polysorbate orpoloxamer surfactant to an etanercept formulation in an amountconstituting about 0.0001 to about 0.5 w/v % (preferably about 0.01 to0.05 w/v %) of the formulation such that the number of subvisibleparticles per ml of the formulation having a size of about 1 to 10microns is reduced by at least 50%, and preferably by at least about 60to 70%, in comparison to the same formulation prepared in the absence ofsaid surfactant; and where the number of sub-visible particles ismeasured by flowcam analysis conducted at the time the formulation isprepared and prior to any appreciable storage or thermal stress thereof.

In a further embodiment, the invention provides an article ofmanufacture comprising containment means containing an etanerceptformulation in final dosage form, wherein the formulation comprisesetanercept and a pharmaceutically acceptable surfactant, and wherein theamount of sub-visible particles present in the formulation issubstantially reduced in comparison to the same formulation lacking thesurfactant. The surfactant, present in the formulation in an amount inthe range of 0.0005 to 1% (w/v), is preferably a polysorbate (e.g.,polysorbate 80) or a polaxamer surfactant (e.g., Pluronic F-68) asreferenced above. If desired, the contained formulation may include oneor more of the further stabilizing ingredients discussed above. Theamount of sub-visible particles in the contained formulation, determinedby conventional means such as flowCAM analysis at “T0”, is preferablysuch that the formulation has (i) less than about 2500 particles/mlhaving a size of about 5-10 μm; (ii) less than about 3500 particleshaving size of about 2-5 μm and (iii) less than about 700 particles/mlhaving size of about 1-2 μm. The level of sub-visible particles presentin the contained etanercept/surfactant formulation preferably representsat least about a 50% to 70% reduction in sub-visible particles incomparison to the same contained formulation having no surfactant.

In yet another embodiment, the invention affords a method for reducing asubject's exposure to sub-visible particles in an etanerceptformulation, said method comprising administering to the subject anetanercept formulation comprising etanercept and a pharmaceuticallyacceptable surfactant, and wherein the amount of sub-visible particlespresent in the formulation is substantially reduced in comparison to thesame formulation lacking the surfactant. The surfactant, present in theformulation in an amount in the range of 0.0005 to 1% (w/v), ispreferably a polysorbate (e.g., polysorbate 80) or a polaxamersurfactant (e.g., Pluronic F-68) as referenced above. If desired, thecontained formulation may include one or more of the further stabilizingingredients discussed above. The amount of sub-visible particles in theformulation, determined by conventional means such as flowCAM analysisat “T0”, is preferably such that the formulation has (i) less than about2500 particles/ml having a size of about 5-10 μm; (ii) less than about3500 particles having size of about 2-5 μm and (iii) less than about 700particles/ml having size of about 1-2 μm. The level of sub-visibleparticles present in the etanercept/surfactant formulation preferablyrepresents at least about a 50% to 70% reduction in sub-visibleparticles in comparison to the same formulation having no surfactant.

Unlike commercially available etanercept, we found it surprising thateach of the formulation embodiments of etanercept described andexemplified herein do not require arginine for long term stabilization,although arginine may still be added if desired. Moreover, with orwithout arginine, and utilizing the surfactants described herein, theformulations are able to exhibit excellent reduction in sub-visibleparticles. The ability to provide etanercept formulations stabilizedwithout arginine, and exhibiting excellent resistance to subvisibleparticle formation, represents a potentially significant benefit to thehealth care system by providing patients and health care providers withalternative formulations of etanercept, including biosimilar andbiobetter variants of commercial Enbrel®, that may become available atlower cost compared with present commercial etanercept formulation(i.e., Enbrel®) that require arginine for stabilization. The reductionis sub-visible particles is also seen as a very significant advantage interms of reducing the immunogenicity of protein therapeutics.

As used herein the term “instability” or like terms denotes the tendencyof the etanercept monomer to undergo a variety of undesiredtransformations during storage, including transformations that mayresult in the formation or presence of subvisible particles of varyingparticle size. Such transformations include the formation of very highmolecular weight aggregate(s) in which multiple copies of theessentially intact etanercept monomer become randomly associated withone another through a variety of non-covalent attractions (e.g.,electrostatic interactions.) Undesired transformations during storagemay also include degradation of the etanercept monomer to smallerfragments and/or oligomers. Ideally, a formulation of etanercept shouldminimize, to the greatest extent possible, the tendency of theformulation to result, during storage, in the formation of subvisibleparticles, aggregates, oligomers and/or fragments of the etanerceptmonomer. An important benefit resulting from the ability to reduceformation of unwanted aggregates or fragments is a reduction in theimmunogenicity of the drug.

Each of the embodiments referenced above may be provided in aformulation which is optionally free, or essentially free of arginine.The term “essentially free of arginine” is intended to mean thatarginine, even if present, is not contributing to the stabilization ofthe etanercept monomer in the formulation to such an extent that aperson skilled in the art would judge its presence beneficial from astabilization standpoint.

These and other aspects will become apparent from the followingdescription of the various embodiments, although variations andmodifications therein may be affected without departing from the spiritand scope of the novel concepts of the disclosure.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a bar chart representing flowCAM analysis for sub-visibleparticles measured at “T0” corresponding to the data in Table VIII ofthe specification. “T0” as explained further in the specificationdenotes evaluation of sub-visible particles at a point in time where thestudied samples have been subject to no appreciable thermal stress orstorage time. The values on the Y axis are particles per ml. In thelegend: F02C is the formulation of Example 4.2 (without PS80); F03C isthe formulation of Example 3.5 (without PS 80); F04C is Enbrel®; F05C isthe formulation of Example 4.2 (with PS80) and F06C is the formulationof Example 3.5 (with PS80). The data in the graph show a markedreduction in sub-visible particles when PS80 is present in theformulations of Examples 4.2 and 3.5, versus the same formulationscontaining no surfactant.

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments of the invention are now described in detail. Asused in the description and throughout the claims, the meaning of “a”,“an”, and “the” includes plural reference unless the context clearlydictates otherwise. Also, as used in the description and throughout theclaims, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise. Additionally, some terms used in thisspecification are more specifically defined below.

Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitionerregarding the description of the invention. Synonyms for certain termsare provided. A recital of one or more synonyms does not exclude the useof other synonyms. The use of examples anywhere in this specificationincluding examples of any terms discussed herein is illustrative only,and in no way limits the scope and meaning of the invention or of anyexemplified term. The invention is not limited to the variousembodiments given in this specification.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In the case of conflict, thepresent document, including definitions will control.

“Around,” “about” or “approximately” shall generally mean within 20percent, within 10 percent, within 5, 4, 3, 2 or 1 percent of a givenvalue or range. Numerical quantities given are approximate, meaning thatthe term “around,” “about” or “approximately” can be inferred if notexpressly stated.

The term “etanercept” or “etanercept monomer” or “monomer” is synonymouswith Enbrel®. It refers to a polypeptide which is a dimeric fusionpolypeptide consisting of the extracellular ligand-binding portion ofthe human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR)linked to the Fc portion of human IgG1. It consists of 934 amino acidsand has an apparent molecular weight of approximately 150 kilodaltons.For the purposes of the present application, the term “etanercept” alsoencompasses etanercept with minor modifications in the amino acidstructure (including deletions, additions, and/or substitutions of aminoacids) which do not significantly affect the function, potency, oravidity of etanercept. The term “etanercept” encompasses all forms andformulations of Enbrel®, including but not limited to concentratedformulations, injectable ready-to-use formulations; formulationsreconstituted with water, alcohol, and/or other ingredients, and others.The term etanercept is also intended to include biosimilar or biobettervariants of the etanercept used in commercial Enbrel®. For example, abiosimilar or biobetter of etanercept may have a slightly differentglycosylation profile than commercial Enbrel®. In addition a biosimilaror bio better variant of the etanercept found in commercial Enbrel® mayexhibit a reduction in the amount of aggregates constituting the activeetanercept ingredient.

The term “monomer” as used herein is intended to mean the dimericetanercept fusion protein referenced above.

The term “meglumine” refers to a compound with chemical formulaH₃NHCH₂(CHOH)₄CH₂OH, also known as 1-Deoxy-1-methylaminosorbitol;N-Methyl-d-glucamine; and 1-Deoxy-1-methylamino-D-glucitol.

The terms “mannosylglycerate,” “mannosyllactate,” “mannosylglycolate”,and “diglycerolphosphate” are well known in the art and have theircommonly accepted meanings. The following references describe thesecompounds in some detail: Faria et al., Carbohydrate Res. 2008, 343:3025-3033; Borges et al., Extremophiles 2002, 6: 209-216; Faria et al.,ChemBioChem 2003, 4: 734-741; Sawangwan et al., Biotechnol. J. 2010, 5:187-191; and Pais et al., J. Mol. Biol. 2009, 394: 237-250. Theapplication incorporates by reference the description of these compoundscontained in these references. The term “serine” refers to an amino acidwhose codons are UCU, UCC, UCA, UCG, AGU, and AGC

The tern “proline” refers to an α-amino acid whose codons are CCU, CCC,CCA, and CCG.

The term “glutamate” refers to a carboxylate anion or salt of glutamicacid (Glu). For the purposes of this application, the term “glutamate”also encompasses glutamic acid itself.

The term “sugar” refers to monosaccharides, disaccharides, andpolysaccharides. Examples of sugars include, but are not limited to,sucrose, glucose, dextrose, and others.

The term “polyol” refers to an alcohol containing multiple hydroxylgroups. Examples of polyols include, but are not limited to, mannitol,sorbitol, and others.

The term “metal ion” refers to a metal atom with a net positive ornegative electric charge. For the purposes of the present application,the term “metal ion” also includes sources of metal ions, including butnot limited to metal salts.

The term “long-term storage” is understood to mean that thepharmaceutical composition can be stored for three months or more, forsix months or more, and preferably for one year or more. Long termstorage is also understood to mean that the pharmaceutical compositionis stored either as a liquid at 2-8° C., or is frozen, e.g., at −20° C.,or colder. It is also contemplated that the composition can be frozenand thawed more than once.

The term “stable” or “stabilized” with respect to long-term storage isunderstood to mean that etanercept contained in the pharmaceuticalcompositions does not lose more than 20%, or more preferably 15%, oreven more preferably 10%, and most preferably 5% of its activityrelative to activity of the composition at the beginning of storage.Stability also denotes a reduced tendency of an etanercept formulationto exhibit sub-visible particles after long term storage.

The term “mammal” includes, but is not limited to, a human.

The term “pharmaceutically acceptable carrier” refers to a non-toxicsolid, semisolid or liquid filler, diluent, encapsulating material,formulation auxiliary, or excipient of any conventional type. Apharmaceutically acceptable carrier is non-toxic to recipients at thedosages and concentrations employed and is compatible with otheringredients of the formulation.

The term “composition” refers to a mixture that usually contains acarrier, such as a pharmaceutically acceptable carrier or excipient thatis conventional in the art and which is suitable for administration intoa subject for therapeutic, diagnostic, or prophylactic purposes. It mayinclude a cell culture in which the polypeptide or polynucleotide ispresent in the cells or in the culture medium. For example, compositionsfor oral administration can form solutions, suspensions, tablets, pills,capsules, sustained release formulations, oral rinses or powders.

The terms “pharmaceutical composition” and “formulation” are usedinterchangeably.

The term “treatment” refers to any administration or application ofremedies for disease in a mammal and includes inhibiting the disease,arresting its development, relieving the disease, for example, bycausing regression, or restoring or repairing a lost, missing, ordefective function; or stimulating an inefficient process. The termincludes obtaining a desired pharmacologic and/or physiologic effect,covering any treatment of a pathological condition or disorder in amammal. The effect may be prophylactic in terms of completely orpartially preventing a disorder or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disorder and/oradverse affect attributable to the disorder. It includes (1) preventingthe disorder from occurring or recurring in a subject who may bepredisposed to the disorder but is not yet symptomatic, (2) inhibitingthe disorder, such as arresting its development, (3) stopping orterminating the disorder or at least its associated symptoms, so thatthe host no longer suffers from the disorder or its symptoms, such ascausing regression of the disorder or its symptoms, for example, byrestoring or repairing a lost, missing or defective function, orstimulating an inefficient process, or (4) relieving, alleviating orameliorating the disorder, or symptoms associated therewith, whereameliorating is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, such as inflammation, pain and/or tumorsize.

The term “disease” refers to any condition, infection, disorder orsyndrome that requires medical intervention or for which medicalintervention is desirable. Such medical intervention can includetreatment, diagnosis and/or prevention.

The term “therapeutically effective amount” refers to an amount which,when administered to a living subject, achieves a desired effect on theliving subject. For example, an effective amount of the polypeptide ofthe invention for administration to the living subject is an amount thatprevents and/or treats an integrin αvβ3-mediated disease. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques. As isknown in the art, adjustments for systemic versus localized delivery,age, body weight, general health, sex, diet, time of administration,drug interaction and the severity of the condition may be necessary, andwill be ascertainable with routine experimentation by those skilled inthe art.

The term “T₁” refers to a point in time at which an etanerceptformulation has been stored for about one week at 40° C.

The term “T₂” refers to a point in time at which an etanerceptformulation has been stored for about two weeks at 40° C.

The term “T₄” refers to a point in time at which an etanerceptformulation has been stored for about four weeks at 40° C.

EMBODIMENTS OF THE INVENTION

When pharmaceutical compositions containing etanercept (Enbrel®),including aqueous and lyophilized formulations of etanercept are storedon a long term basis, the activity of etanercept can be lost ordecreased due to instability of the etanercept monomer via aggregationand/or degradation including formation of fragments and oligomers.Moreover, the formulation may exhibit sub-visible particles over time.Thus, the present invention provides several embodiments of aqueousformulations of etanercept that allow stable long-term storage ofetanercept, and reduction in sub-visible particles, so that etanerceptis stable over the course of storage either in liquid or frozen states.The provided formulations include, but are not limited to formulationswhich do not contain arginine and do not require any extra steps such asrehydrating.

These embodiments are explained in a greater detail below.

Etanercept

All of the compositions of the present invention contemplate the use ofetanercept including biosimilar or biobetter variants of the etanerceptused in commercial Enbrel®. As explained in the Background section ofthis application, etanercept is a dimeric fusion polypeptide consistingof the extracellular ligand-binding portion of the human 75 kilodalton(p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion ofhuman IgG1. Etanercept consists of 934 amino acids. The Fc component ofetanercept contains the constant heavy 2 (CH2) domain, the constantheavy 3 (CH3) domain and hinge region of human IgG1. An Fc domain cancontain one or all of the domains described above.

Etanercept suitable for storage in the present pharmaceuticalcomposition can be produced by living host cells that expressetanercept, such as hybridomas in the case of antibodies, or host cellsthat that have been genetically engineered to produce the polypeptide inthe case of fusion polypeptides or antibodies. Methods of geneticallyengineering cells to produce polypeptides are well known in the art.See, e.g., Ausubel et al., eds. (1990), Current Protocols in MolecularBiology (Wiley, New York). Such methods include introducing nucleicacids that encode and allow expression of the polypeptide into livinghost cells. These host cells can be bacterial cells, fungal cells, or,preferably, animal cells grown in culture. Bacterial host cells include,but are not limited to, Escherichia coli cells. Examples of suitable E.coli strains include: HB101, DH5.alpha, GM2929, JM109, KW251, NM538,NM539, and any E. coli strain that fails to cleave foreign DNA. Fungalhost cells that can be used include, but are not limited to,Saccharomyces cerevisiae, Pichia pastoris and Aspergillus cells. A fewexamples of animal cell lines that can be used are CHO, VERO, BHK, HeLa,Cos, MDCK, 293, 3T3, and W138. New animal cell lines can be establishedusing methods well know by those skilled in the art (e.g., bytransformation, viral infection, and/or selection). Optionally,etanercept can be secreted by the host cells into the medium.

Purification of the expressed etanercept can be performed by anystandard method. When etanercept is produced intracellularly, theparticulate debris is removed, for example, by centrifugation orultrafiltration. When etanercept is secreted into the medium,supernatants from such expression systems can be first concentratedusing standard polypeptide concentration filters. Protease inhibitorscan also be added to inhibit proteolysis and antibiotics can be includedto prevent the growth of microorganisms.

Etanercept can be purified using, for example, hydroxyapatitechromatography, gel electrophoresis, dialysis, and affinitychromatography, and any combination of known or yet to be discoveredpurification techniques, including but not limited to Protein Achromatography, fractionation on an ion-exchange column, ethanolprecipitation, reverse phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSET®, an anion or cation exchange resinchromatography (such as a polyaspartic acid column), chromatofocusing,SDS-PAGE, and ammonium sulfate precipitation.

I. Etanercept Stabilized with Surfactant, Plus Serine, Proline orGlutamate

In one embodiment, the invention provides a stable aqueouspharmaceutical composition comprising etanercept, a polysorbate orpoloxamer surfactant, and a stabilizer to inhibit instability,aggregation and/or fragmentation of the etanercept, wherein thestabilizer comprises a compound selected from the group consisting ofserine, proline and glutamate. In a preferred embodiment, the stabilizercomprises glutamate.

Without intending to be bound to any particular theory of the invention,it is believed that serine, proline and glutamate act as stabilizers toreduce etanercept's tendency to associate in undesired ternary orquaternary complexes, and therefore to reduce aggregation of etanercept.The reduction in aggregation is believed to last for a long period oftime, e.g., two years or more. It is believed that serine, proline andglutamate are able to stabilize aqueous pharmaceutical compositionscontaining etanercept because they are excluded from the surface of theprotein, resulting in net conformation stabilization. The stabilizingeffects of serine, proline and/or glutamate include but are not limitedto the benefits of reduced aggregation of the etanercept monomer informulations containing the monomer.

The pharmaceutical compositions of the invention may be prepared bycombining a purified etanercept and the stated stabilizers. Further, abuffer, a tonicity modifier and an additional excipient and othercommonly used inactive ingredients can be added as needed. Forsimplicity, these are discussed more fully later in the specification. Aperson of ordinary skill in the art will understand that the combiningof the various components to be included in the composition can be donein any appropriate order. For example, the buffer can be added first,middle or last, and the tonicity modifier can also be added first,middle or last. A person of ordinary skill in the art will alsounderstand that some of these chemicals can be incompatible in certaincombinations, and accordingly, are easily substituted with differentchemicals that have similar properties but are compatible in therelevant mixture.

In a preferred embodiment, the concentration of the serine, proline orglutamate stabilizer in the provided formulations is preferably up toabout 150 mM.

Serine, proline and glutamate are available from commercial suppliers.

In an embodiment in which the stabilizer comprises surfactant andglutamate, a formulation of the invention can comprise about 0.01 toabout 0.05% w/v of the polysorbate or poloxamer surfactant; about 25 toabout 50 mg/ml of etanercept; up to 150 mM glutamate; less than about 6wt % sucrose; optionally up to about 100 mM NaCl; about 1 to about 30 mMsodium phosphate, and wherein the formulation has pH 6.0 to about pH 7.0and more preferably about 6.0 to about 6.6, and most preferably betweenabout 6.3 to about 6.5.

In an embodiment in which the stabilizer comprises surfactant andserine, a formulation of the invention can comprise about 0.01 to about0.05% w/v of the polysorbate or poloxamer surfactant; about 25 to about50 mg/ml of etanercept; less than about 150 mM serine; about 0.5 toabout 3 wt % sucrose; about 1 to about 30 mM sodium phosphate, andwherein the formulation has pH 6.0 to about pH 7.0 and more preferablyabout 6.0 to about 6.6, and most preferably between about 6.3 to about6.5.

In an embodiment in which the stabilizer comprises proline, aformulation of the invention can comprise about 0.01 to about 0.05% w/vof the polysorbate or poloxamer surfactant; about 25 to about 50 mg/mlof etanercept; less than about 150 mM proline; about 0.5 to about 3 wt %sucrose; about 1 to about 30 mM sodium phosphate, about 15 to about 100mM NaCl; and wherein the formulation has pH 6.0 to about pH 7.0 and morepreferably about 6.0 to about 6.6, and most preferably between about 6.3to about 6.5.

Etanercept formulations according to the present invention comprisingserine, proline or glutamate are preferably characterized by an SECanalysis at T₂ of: about 80 to about 95 wt % monomer content; less thanabout 4 wt % aggregate(s) content; and less than about 8 wt % fragment 3content.

In formulations containing serine, proline or glutamate forstabilization, the formulations are more preferably characterized by:

-   -   (a) an SEC analysis at T₄ of greater than about 90, 91, 92, 93,        94, 95, 96, or 97 wt % monomer content; and less than about 3, 2        or 1 wt % aggregate(s) content; and    -   (b) an HIC analysis at T₂ wherein the amount of the composition        represented by peak 1 of the HIC chromatogram is less than about        3, 2 or 1 wt %; the amount of the composition represented by        peak 2 of the HIC chromatogram is greater than 80, 81, 82, 83,        84 or 85 wt %; and the amount of the composition represented by        peak 3 of the HIC chromatogram is less than about 20, 19, 18,        17, 16, 15, 14, or 13 wt %; and    -   (c) an HIC analysis at T₄ wherein the amount of the composition        represented by peak 1 of the HIC chromatogram is less than about        3, 2 or 1 wt %; the amount of the composition represented by        peak 2 of the HIC chromatogram is greater than 80, 81, 82, 83,        84 or 85 wt %; and the amount of the composition represented by        peak 3 of the HIC chromatogram is less than 20, 19, 18, 17, 16,        15, 14 or 13 wt %.

The terms “SEC”, “T₂” “T₄” “HIC” “monomer content” “aggregate(s)” and“fragment 3” “peak 1,” “peak 2,” and “peak 3,” are defined in theexamples below.

In particularly preferred formulations containing surfactant plusserine, proline or glutamate for stabilization preferably arecharacterized by having an HIC analysis at T₄ or T₂ wherein the amountof the composition represented by peak 1 of the HIC chromatogram is lessthan about 1%; the amount of the composition represented by peak 2 ofthe HIC chromatogram is greater than about 95 wt. %, and most preferablygreater than about 99 wt %; and the amount of the compositionrepresented by peak 3 of the HIC chromatogram is less than about 1 wt %.

A further preferred formulation using surfactant plus serine, prolineand/or glutamate for stabilization of etanercept comprises about 50mg/ml etanercept; less than about 150 mM serine, proline or glutamate,and most preferably glutamate; about 0 to 3% sucrose; about 1 to 30 mMphosphate buffer, and having a pH of about 6.0 to 6.6; and characterizedby: an SEC analysis at T₄ of greater than about 97 wt. % monomer contentand less than about 1 wt % aggregate(s) content; an HIC analysis at T₂wherein the amount of the composition represented by peak 1 of the HICchromatogram is less than about 3 wt %; the amount of the compositionrepresented by peak 2 of the HIC chromatogram is greater than about 82wt %; and the amount of the composition represented by peak 3 of the HICchromatogram is less than about 15 wt %; and an HIC analysis at T₄wherein the amount of the composition represented by peak 1 of the HICchromatogram is less than about 2 wt %; the amount of the compositionrepresented by peak 2 of the HIC chromatogram is greater than about 84wt %; and the amount of the composition represented by peak 3 of the HICchromatogram is less than about 13 wt %.

A further preferred glutamate stabilized etanercept formulationcomprises: about 0.01 to about 0.05% w/v of the polysorbate or poloxamersurfactant; about 50 mg/ml etanercept; about 120 mM glutamate; about 1%sucrose, and about 25 mM phosphate; having a pH of about 6.3 to about6.5, and exhibiting the SEC and HIC analytical characteristicsreferenced above.

Although the invention does not exclude the use of arginine, theetanercept formulations comprising about 0.01 to about 0.05% w/v of thepolysorbate or poloxamer surfactant; plus serine, proline and/orglutamate for additional stabilization according to the presentinvention are preferably free or essentially free of arginine.

II. Etanercept Stabilized with Surfactant Plus a Metal Ion

In another embodiment, the invention provides a stabilized aqueouspharmaceutical composition comprising etanercept, about 0.01 to about0.05% w/v of the polysorbate or poloxamer surfactant; and a stabilizerto inhibit instability, aggregation and/or fragmentation of theetanercept, wherein the stabilizer comprises a stabilizing metal ion.

It is believed that metal ions such as calcium, magnesium, and zincreduce etanercept's tendency to associate in undesired ternary orquaternary complexes, and therefore, reduce aggregation of etanercept.The reduction in aggregation is believed to last for a long period oftime, e.g., two years or more. Without wishing to be bound to aparticular theory, it is believed that metal ions are able to stabilizeaqueous pharmaceutical compositions containing etanercept because themetal can bind to the native state, where the right geometry of ligandsoccurs. In doing so, there is a net stabilization of the native state.Once the protein unfolds, the binding site is lost, and the denaturedstate in relatively unaffected in terms of free energy. The result is anet stabilization of the conformation, leading to improved long-termstorage. In addition, metal biding may also improve the colloidalstability of the protein, elading to decreased aggregation and increasedsolubility. The stabilization effects of metal ion are may not belimited to reduction in aggregates but may also address other aspects ofinstability of the etanercept monomer in the formulation.

In a preferred embodiment, the metal ion is selected from the groupconsisting of calcium, magnesium, zinc, and combinations thereof. In aneven more preferred embodiment, calcium, magnesium, and zinc areprovided as calcium chloride, magnesium chloride and zinc chloride,respectively.

The pharmaceutical compositions of the invention may be prepared bycombining, a purified etanercept, about 0.01 to about 0.05% w/v of thepolysorbate or poloxamer surfactant; and a metal ion. Further, a buffer,a tonicity modifier and an additional excipient and other commonly usedinactive ingredients can be added as needed. For simplicity, these arediscussed more fully later in the specification. A person of ordinaryskill in the art will understand that the combining of the variouscomponents to be included in the composition can be done in anyappropriate order. For example, the buffer can be added first, middle orlast, and the tonicity modifier can also be added first, middle or last.A person of ordinary skill in the art will also understand that some ofthese chemicals can be incompatible in certain combinations, andaccordingly, are easily substituted with different chemicals that havesimilar properties but are compatible in the relevant mixture.

In a preferred embodiment, the concentration of the metal ion in theprovided formulations is preferably between about 1 mM to 0.5 M, morepreferably about 1 mM to about 100 mM, more preferably about 2 mM toabout 20 mM, and yet more preferably about 2 to 10 mM.

Sources of metal ions are available from commercial suppliers.

In an embodiment using surfactant plus calcium chloride forstabilization, an etanercept formulation of the invention comprisesabout 0.01 to about 0.05% w/v of the polysorbate or poloxamersurfactant; about 25 to about 50 mg/ml of etanercept; up to about 5 mMcalcium chloride; optionally about 0.5 to 6 wt % sucrose or trehalose;optionally about 0 to 100 mM NaCl; optionally up to about 10 mM xylitol;about 1 to about 30 mM sodium phosphate; wherein the composition has apH of about 6.0 to about pH 7.0, and more preferably about 6.0 to about6.6 and most preferably about 6.3 to about 6.5.

In an embodiment using magnesium chloride for stabilization, anetanercept formulation of the invention comprises about 0.01 to about0.05% w/v of the polysorbate or poloxamer surfactant; about 25 to about50 mg/ml of etanercept; about 1 mM to about 20 mM magnesium chloride;optionally up to about 6 wt % sucrose; about 25 to 150 mM NaCl; about 1to about 30 mM sodium phosphate; wherein the composition has a pH ofabout 6.0 to about pH 7.0, and more preferably about 6.0 to about 6.6and most preferably about 6.3 to about 6.5.

Compositions stabilized with metal ions are preferably characterized ashaving an SEC analysis at T₂ of: about 80 wt % to about 95 wt % monomercontent; an SEC analysis at T₂ of aggregate(s) content of less thanabout 4 wt %; and an SEC analysis at T₂ of fragment 3 content of lessthan about 8 wt %.

More preferably the etanercept formulations containing a stabilizingmetal ion according to the invention are characterized by:

-   -   (a) an SEC analysis at T₄ of greater than about 90, 91, 92, 93,        94, 95, 96, or 97 wt % monomer content; and less than about 3, 2        or 1 wt % aggregate(s) content; and    -   (b) an HIC analysis at T₂ wherein the amount of the composition        represented by peak 1 of the HIC chromatogram is less than about        3, 2 or 1 wt %; the amount of the composition represented by        peak 2 of the HIC chromatogram is greater than 80, 81, 82, 83,        84 or 85 wt %; and the amount of the composition represented by        peak 3 of the HIC chromatogram is less than about 20, 19, 18,        17, 16, 15, 14, or 13 wt %; and    -   (c) an HIC analysis at T₄ wherein the amount of the composition        represented by peak 1 of the HIC chromatogram is less than about        3, 2 or 1 wt %; the amount of the composition represented by        peak 2 of the HIC chromatogram is greater than 80, 81, 82, 83,        84 or 85 wt %; and the amount of the composition represented by        peak 3 of the HIC chromatogram is less than 20, 19, 18, 17, 16,        15, 14 or 13 wt %.

The etanercept formulations of the present invention containingsurfactant plus metal ion for stabilization are more preferablycharacterized by having an HIC analysis at T₄ or T₂ wherein the amountof the composition represented by peak 1 of the HIC chromatogram is lessthan about 1%; the amount of the composition represented by peak 2 ofthe HIC chromatogram is greater than about 95 wt % and most preferablygreater than about 99 wt %; and the amount of the compositionrepresented by peak 3 of the HIC chromatogram is less than about 1 wt %.

The terms “SEC”, “T₂” “T₄” “HIC” “monomer content” “aggregate(s)” and“fragment 3” “peak 1,” “peak 2,” and “peak 3,” are defined in theexamples below.

Preferred etanercept formulations stabilized with surfactant pluscalcium chloride comprise: about 0.01 to about 0.05% w/v of thepolysorbate or poloxamer surfactant; about 50 mg/ml etanercept; 1 to 5mM calcium chloride; about 1 to 30 mM sodium phosphate; about 0 to 100mM NaCl; about 0.5 to 5% sucrose or trehalose or combination thereof;and wherein the composition has a pH of about 6.0 to 6.6 andcharacterized by: an SEC analysis at T₄ of greater than about 97 wt. %monomer content and less than about 1 wt % aggregate(s) content; an HICanalysis at T₂ wherein the amount of the composition represented by peak1 of the HIC chromatogram is less than about 4 wt %; the amount of thecomposition represented by peak 2 of the HIC chromatogram is greaterthan about 82 wt %; and the amount of the composition represented bypeak 3 of the HIC chromatogram is less than about 15 wt %; and an HICanalysis at T₄ wherein the amount of the composition represented by peak1 of the HIC chromatogram is less than about 2 wt %; the amount of thecomposition represented by peak 2 of the HIC chromatogram is greaterthan about 85 wt %; and the amount of the composition represented bypeak 3 of the HIC chromatogram is less than about 13 wt %.

Preferred etanercept formulations stabilized with surfactant plusmagnesium chloride comprise: about 0.01 to about 0.05% w/v of thepolysorbate or poloxamer surfactant; about 1 mM to about 20 mM magnesiumchloride; optionally up to about 6 wt % sucrose; about 25 to 150 mMNaCl; about 1 to about 30 mM sodium phosphate; wherein the compositionhas a pH of about 6.0 to 6.6; and wherein the composition ischaracterized by: an SEC analysis at T₄ of greater than about 97 wt. %monomer content and less than about 1 wt % aggregate(s) content; an HICanalysis at T₂ wherein the amount of the composition represented by peak1 of the HIC chromatogram is less than about 4 wt %; the amount of thecomposition represented by peak 2 of the HIC chromatogram is greaterthan about 85 wt %; and the amount of the composition represented bypeak 3 of the HIC chromatogram is less than about 14 wt %; and an HICanalysis at T₄ wherein the amount of the composition represented by peak1 of the HIC chromatogram is less than about 2 wt %; the amount of thecomposition represented by peak 2 of the HIC chromatogram is greaterthan about 85 wt %; and the amount of the composition represented bypeak 3 of the HIC chromatogram is less than about 14 wt %.

In particularly preferred embodiments of the invention using surfactantplus calcium chloride for stabilization, a stabilized etanerceptformulation having the analytical properties referenced above comprises:about 50 mg/ml of etanercept; about 2 mM calcium chloride; about 15 mMsodium phosphate; about 75 mM sodium chloride; and about 3 wt % sucrose;wherein the formulation has a pH of about 6.3 to 6.5.

In a further preferred embodiment of the invention using surfactant plusmagnesium chloride for stabilization, a stabilized etanerceptformulation having the analytical properties referenced above comprises:about 0.01 to about 0.05% w/v of the polysorbate or poloxamersurfactant; about 50 mg/ml of etanercept; about 10 mM magnesiumchloride; about 15 mM sodium phosphate; about 75 mM sodium chloride; andabout 3 wt % sucrose; and having a pH of about 6.3 to 6.5.

The terms “SEC”, “T₂” “T₄” “HIC” “monomer content” “aggregate(s)” and“fragment 3” “peak 1,” “peak 2,” and “peak 3,” are defined in theexamples below.

Although the use of surfactant plus stabilizing metal ions according tothe invention does not exclude the use of arginine, the etanerceptformulations comprising metal ion for stabilization according to thepresent invention are preferably free or essentially free of arginine.

III. Etanercept Stabilized with Surfactant Plus an Ionic PolyolDerivative Excipient

In another embodiment, the invention provides a stable aqueousformulation comprising etanercept, about 0.01 to about 0.05% w/v of thepolysorbate or poloxamer surfactant; and an ionic polyol derivativeexcipient, wherein said excipient is selected from the group consistingof meglumine (N-methyl-D-glucamine), mannosylglycerate,glucosylglycerate, mannosyllactate, mannosylglycolate, anddiglycerolphosphate.

Preferably, in this embodiment or aspect, the invention is an aqueousstabilized formulation of etanercept comprising: etanercept; about 0.01to about 0.05% w/v of the polysorbate or poloxamer surfactant; andstabilizing ingredients to retard instability, aggregation andfragmentation of the etanercept in the formulation, said stabilizingingredients being comprised of (a) meglumine; or (b) meglumine incombination with sucrose; or (c) meglumine in combination with sodiumchloride; or (d) meglumine in combination with sodium chloride andsucrose.

Meglumine is commonly used as a small molecule excipient. We have nowsurprisingly found that meglumine is also able to stabilize aqueouspharmaceutical compositions containing a large protein, such asetanercept.

It is believed that meglumine reduces etanercept's tendency to associatein undesired ternary or quaternary complexes, and therefore, reducesaggregation of etanercept. The reduction in aggregation is believed tolast for a long period of time, e.g., two years or more. Without wishingto be bound to a particular theory, it is believed that meglumine isable to stabilize aqueous pharmaceutical compositions containingetanercept by a combination of three different mechanisms. First,meglumine can act as an excluded solute in the same way mannitol,sucrose, and sorbitol increase conformational stability. Second, chargedsolutes can alter the colloidal stability, thereby reducing thepropensity to self-associate, thereby slowing aggregation. Third, theseionic polyol derivatives, being charged near neutral pH, can act assalting-in agents, as arginine does, potentially resolubilizingaggregates. The stabilizing effects of meglumine are not limited toreduction in aggregates but may involve other aspects of stabilizationof the etanercept monomer in a formulation containing the monomer.

The pharmaceutical compositions of the invention may be prepared bycombining, a purified etanercept, about 0.01 to about 0.05% w/v of thepolysorbate or poloxamer surfactant, and the ionic polyol derivative,preferably meglumine. Further, a buffer, a tonicity modifier and anadditional excipient and other commonly used inactive ingredients can beadded as needed. For simplicity, these are discussed more fully later inthe specification. A person of ordinary skill in the art will understandthat the combining of the various components to be included in thecomposition can be done in any appropriate order. For example, thebuffer can be added first, middle or last, and the tonicity modifier canalso be added first, middle or last. A person of ordinary skill in theart will also understand that some of these chemicals can beincompatible in certain combinations, and accordingly, are easilysubstituted with different chemicals that have similar properties butare compatible in the relevant mixture.

In a preferred embodiment, the concentration of meglumine in theprovided formulations is preferably between about 0.1% (w/v) to 40%(w/v), more preferably about 1% to about 20%, more preferably about 2%to about 10%, even more preferably about 2% to about 5%.

Meglumine is available from commercial suppliers.

A preferred embodiment comprises about 25 to about 75 mg/ml etanercept,about 0.01 to about 0.05% w/v of the polysorbate or poloxamersurfactant; about 1-30 mM of sodium phosphate; up to about 10%meglumine; optionally up to about 5 wt % sucrose; and optionally up toabout 100 mM sodium chloride, wherein the composition has a pH of about6.0 to 7.0, and preferably about 6.0 to about 6.6 and most preferablyabout 6.3 to about 6.5.

A surfactant/meglumine stabilized etanercept composition is preferablycharacterized by SEC analysis at T₂ in which: the monomer content isgreater than about 85 wt. %; aggregate(s) content is less than about 3wt %; and fragment 3 content is less than about 8 wt. %.

A more preferred formulation of etanercept wherein an ionic polyolderivative such as meglumine is present for stabilization is one that ischaracterized by:

-   -   (a) an SEC analysis at T₄ of greater than about 90, 91, 92, 93,        94, 95, 96, or 97 wt % monomer content; and less than about 3, 2        or 1 wt % aggregate(s) content; and    -   (b) an HIC analysis at T₂ wherein the amount of the composition        represented by peak 1 of the HIC chromatogram is less than about        4, 3, 2 or 1 wt %; the amount of the composition represented by        peak 2 of the HIC chromatogram is greater than 80, 81, 82, 83,        84 or 85 wt %; and the amount of the composition represented by        peak 3 of the HIC chromatogram is less than about 20, 19, 18,        17, 16, 15, 14, or 13 wt %; and    -   (c) an HIC analysis at T₄ wherein the amount of the composition        represented by peak 1 of the HIC chromatogram is less than about        3, 2 or 1 wt %; the amount of the composition represented by        peak 2 of the HIC chromatogram is greater than 80, 81, 82, 83,        84 or 85 wt %; and the amount of the composition represented by        peak 3 of the HIC chromatogram is less than 20, 19, 18, 17, 16,        15, 14 or 13 wt %.

A particularly preferred etanercept formulation stabilized withsurfactant plus meglumine is characterized by HIC analysis at T₄ or T₂wherein the amount of the composition represented by peak 1 of the HICchromatogram is less than about 1%; the amount of the compositionrepresented by peak 2 of the HIC chromatogram is greater than about 99wt %; and the amount of the composition represented by peak 3 of the HICchromatogram is less than about 1 wt %.

The terms “SEC”, “T₂” “T₄” “HIC” “monomer content” “aggregate(s)” and“fragment 3” “peak 1,” “peak 2,” and “peak 3,” are defined in theexamples below.

In other embodiments meglumine can be replaced with another ionic polyolderivative of sorbitol, glycerol or mannitol, such as mannosylglycerate,glucosylglycerate, mannosyllactate, mannosylglycolate, anddiglycerolphosphate (at about 0.1% to about 40%) in the formulation.

A preferred meglumine-stabilized etanercept formulation containing about0.01 to about 0.05% w/v of the polysorbate or poloxamer surfactant, andfree of arginine and exhibiting analytical properties as described abovecomprises about 25 to about 75 mg/ml etanercept; about 0.5 wt. %meglumine; about 25 mM phosphate; about 1% sucrose; and about 100 mMsodium chloride.

A further preferred surfactant/meglumine-stabilized etanerceptformulation free of arginine and exhibiting analytical properties asdescribed above comprises about 0.01 to about 0.05% w/v of thepolysorbate or poloxamer surfactant; about 50 mg/ml etanercept; about 5wt. % meglumine; about 25 mM phosphate.

Although the invention does not exclude the use of arginine, theetanercept formulations comprising ionic polyol derivatives such asmeglumine for stabilization according to the present invention arepreferably free or essentially free of arginine.

IV. Etanercept Stabilized with Surfactant and a Combination of a Sugarand a Polyol

In yet another embodiment, the invention provides a stable aqueousformulation comprising etanercept, about 0.01 to about 0.05% w/v of thepolysorbate or poloxamer surfactant; a sugar and a polyol.

It is believed that a combination of a sugar and a polyol reducesetanercept's tendency to associate in undesired ternary or quaternarycomplexes, and therefore, reduces aggregation of etanercept. Thereduction in aggregation is believed to last for a long period of time,e.g., two years or more. Thus, a combination of a sugar and a polyol isbelieved to be able to stabilize aqueous pharmaceutical compositionscontaining etanercept. Without wishing to be bound to a particulartheory, the combination of a sugar and a polyol is believed to besynergistic for the purposes of stabilizing etanercept because eventhough excluded solutes are, on average, residing in the bulk, ratherthan on the surface of the protein, the fact is that there will beinteractions between sugars/polyols and the protein. Those interactionswill likely differ between sugars and smaller polyols. In addition, athigh concentrations, the two additives will alter the thermodynamicactivity of the other, thereby leading to solution behavior that will bedifferent than what would be observed for each individual component. Asdiscussed further below, amines can be substituted for the polyol.

The pharmaceutical compositions of the invention may be prepared bycombining, a purified etanercept, about 0.01 to about 0.05% w/v of thepolysorbate or poloxamer surfactant; a sugar, and a polyol. Further, abuffer, a tonicity modifier and an additional excipient and othercommonly used inactive ingredients can be added as needed. Forsimplicity, these are discussed more fully later in the specification. Aperson of ordinary skill in the art will understand that the combiningof the various components to be included in the composition can be donein any appropriate order. For example, the buffer can be added first,middle or last, and the tonicity modifier can also be added first,middle or last. A person of ordinary skill in the art will alsounderstand that some of these chemicals can be incompatible in certaincombinations, and accordingly, are easily substituted with differentchemicals that have similar properties but are compatible in therelevant mixture.

In some embodiments, a sugar and a polyol may act in concert, in thesame way two metals form an alloy with properties not exhibited byeither metal. It should be understood that the same approach would leadone to use amino acids, such as proline, serine, or glutamate along witha sugar to achieve a stability profile better than either excipientcould provide on its own. A preferred ratio of a sugar to a polyol (oramino acid) in the alloy is believed to be between 5:1 to 1:5.

The most preferred sugars are believed to be sucrose, trehalose,lactose, raffinose, and maltose.

The most preferred polyols are believed to be sorbitol, mannitol,glycerol, and propylene glycol.

The preferred amino acids are believed to be proline, serine, threonine,and glutamate.

In a preferred embodiment, the concentration of a sugar in the providedformulations is preferably between about 0.1% (w/v) to 40%, morepreferably about 1% to about 20%, more preferably about 2% to about 10%,and yet more preferably about 5% to 9%.

In a preferred embodiment, the concentration of a polyol in the providedformulations is preferably between about 0.1% to 30%, more preferablyabout 1% to about 10%, and yet more preferably about 2% to about 5%.

Sugars and polyols are available from commercial suppliers.

In one embodiment, a formulation of the invention comprises about 25 toabout 75 mg/ml of etanercept; about 0.01 to about 0.05% w/v of thepolysorbate or poloxamer surfactant; about 1% to about 10% sucrose;about 1% to about 5% mannitol; about 10 mM to about 50 mM sodiumphosphate; and about 0 mM to about 100 mM NaCl, at about pH 6.3 to aboutpH 7.0.

In another embodiment, sucrose can be replaced with another sugar suchas trehalose (at about 1% to about 10%) in the formulation. In yetanother embodiment, mannitol can be replaced with another polyol such assorbitol (at about 1% to about 5%) in the formulation.

Although the invention does not exclude the use of arginine theetanercept formulations comprising sugar and polyol (or amino acid) forstabilization are preferably free or essentially free of arginine.

V. Etanercept Stabilized with Surfactant Plus Xylitol

In yet another embodiment, the invention provides a stabilized aqueouspharmaceutical composition comprising etanercept, about 0.01 to about0.05% w/v of the polysorbate or poloxamer surfactant; and a stabilizerto inhibit instability, aggregation and/or fragmentation of theetanercept, wherein the stabilizer comprises xylitol or a combination ofxylitol and meglumine.

Without wishing to be bound to any particular theory, It is believedthat xylitol reduces etanercept's tendency to associate in undesiredternary or quaternary complexes, and therefore, reduces aggregation ofetanercept. The reduction in aggregation is believed to last for a longperiod of time, e.g., two years or more. The stabilizing effects ofxylitol are not limited to reduction in aggregates but may involve otheraspects of stabilization of the etanercept monomer in a formulationcontaining the monomer.

A preferred stabilized etanercept formulation incorporating xylitol forstabilization is one in which stabilization is provided by about 0.01 toabout 0.05% w/v of the polysorbate or poloxamer surfactant; plus acombination of xylitol and meglumine.

The pharmaceutical compositions of the invention may be prepared bycombining, a purified etanercept, about 0.01 to about 0.05% w/v of thepolysorbate or poloxamer surfactant; and xylitol, or xylitol incombination with meglumine. Further, a buffer, a tonicity modifier andan additional excipient and other commonly used inactive ingredients canbe added as needed. For simplicity, these are discussed more fully laterin the specification. A person of ordinary skill in the art willunderstand that the combining of the various components to be includedin the composition can be done in any appropriate order. For example,the buffer can be added first, middle or last, and the tonicity modifiercan also be added first, middle or last. A person of ordinary skill inthe art will also understand that some of these chemicals can beincompatible in certain combinations, and accordingly, are easilysubstituted with different chemicals that have similar properties butare compatible in the relevant mixture.

Xylitol stabilized etanercept formulations of the invention can compriseabout 25 to 75 mg/ml of etanercept; about 0.01 to about 0.05% w/v of thepolysorbate or poloxamer surfactant; about 1-10 wt. % xylitol; about 1to 30 mM sodium phosphate; optionally up to about 5 wt % meglumine;optionally up to about 5 mM NaCl; and optionally up to about 5 wt %sucrose.

Surfactant/Xylitol stabilized etanercept formulations which additionallycontain meglumine, sodium chloride and sucrose can comprise, in additionto xylitol, comprise about 1-3 mM NaCl; about 1 to 5 wt % sucrose; andmeglumine in an amount of about 1-5 wt. % of the composition.

In a further embodiment, xylitol stabilized etanercept formulations cancomprise about 25 to about 75 mg/ml of etanercept; about 0.01 to about0.05% w/v of the polysorbate or poloxamer surfactant; and a stabilizerto inhibit instability, aggregation and/or fragmentation of theetanercept, wherein the stabilizer is xylitol in an amount constitutingup to about 10 wt. % of the composition, and wherein the composition ischaracterized by an SEC analysis at T₂ of: about 80 wt % to about 95 wt% monomer content; an SEC analysis at T₂ of aggregate(s) content of lessthan about 4 wt % and preferably less than about 3 wt %; and an SECanalysis at T₂ of fragment 3 content of less than about 8 wt % andpreferably less than about 6 wt %; wherein the composition has a pH ofabout 6.0 to about pH 7.0, and more preferably about 6.0 to about 6.6and most preferably about 6.3 to about 6.5.

In stabilized etanercept formulations such as those referenced abovecontaining surfactant plus xylitol or surfactant plus xylitol incombination with meglumine, the formulations are more preferablycharacterized by:

-   -   (a) an SEC analysis at T₄ of greater than about 90, 91, 92, 93,        94, 95, 96, or 97 wt % monomer content; and less than about 3, 2        or 1 wt % aggregate(s) content; and    -   (b) an HIC analysis at T₂ wherein the amount of the composition        represented by peak 1 of the HIC chromatogram is less than about        3, 2 or 1 wt %; the amount of the composition represented by        peak 2 of the HIC chromatogram is greater than 80, 81, 82, 83,        84 or 85 wt %; and the amount of the composition represented by        peak 3 of the HIC chromatogram is less than about 20, 19, 18,        17, 16, 15, 14, or 13 wt %; and    -   (c) an HIC analysis at T₄ wherein the amount of the composition        represented by peak 1 of the HIC chromatogram is less than about        3, 2 or 1 wt %; the amount of the composition represented by        peak 2 of the HIC chromatogram is greater than 80, 81, 82, 83,        84 or 85 wt %; and the amount of the composition represented by        peak 3 of the HIC chromatogram is less than 20, 19, 18, 17, 16,        15, 14 or 13 wt %.

The terms “SEC”, “T₂” “T₄” “HIC” “monomer content” “aggregate(s)” and“fragment 3” “peak 1,” “peak 2,” and “peak 3,” are defined in theexamples below.

Particularly preferred formulations containing about 0.01 to about 0.05%w/v of the polysorbate or poloxamer surfactant; pus xylitol, or xylitolin combination with meglumine, are characterized by having an HICanalysis at T₄ or T₂ wherein the amount of the composition representedby peak 1 of the HIC chromatogram is less than about 1%; the amount ofthe composition represented by peak 2 of the HIC chromatogram is greaterthan about 95 wt. % and preferably greater than about 99 wt %; and theamount of the composition represented by peak 3 of the HIC chromatogramis less than about 1 wt %. Specific xylitol-stabilized formulations areprovided in the detailed examples.

Although the invention does not exclude the use of arginine, theetanercept formulations comprising surfactant plus xylitol forstabilization according to the present invention are free or essentiallyfree of arginine.

VI. Etanercept Formulations Stabilized with Surfactant Plus NaCl

In yet another embodiment, the invention provides an aqueous etanerceptformulation stabilized to reduce instability, aggregation and/orfragmentation of the etanercept, said formulation comprising about 25 toabout 75 mg/ml of etanercept about 0.01 to about 0.05% w/v of thepolysorbate or poloxamer surfactant; and one or more stabilizers,wherein the stabilizers are selected from the group consisting of (i)sodium chloride and (ii) sodium chloride in combination with sucrose ortrehalose; and (iii) a combination of sodium chloride, sucrose andtrehalose.

The pharmaceutical compositions of the invention may be prepared bycombining, a purified etanercept, about 0.01 to about 0.05% w/v of thepolysorbate or poloxamer surfactant; and sodium choride, optionally withsucrose and/or trehalose. Further, a buffer, a tonicity modifier and anadditional excipient and other commonly used inactive ingredients can beadded as needed. For simplicity, these are discussed more fully later inthe specification. A person of ordinary skill in the art will understandthat the combining of the various components to be included in thecomposition can be done in any appropriate order. For example, thebuffer can be added first, middle or last, and the tonicity modifier canalso be added first, middle or last. A person of ordinary skill in theart will also understand that some of these chemicals can beincompatible in certain combinations, and accordingly, are easilysubstituted with different chemicals that have similar properties butare compatible in the relevant mixture.

In an embodiment using surfactant plus sodium chloride forstabilization, an etanercept formulation of the invention comprisesabout 25 to 75 mg/ml etanercept, about 0.01 to about 0.05% w/v of thepolysorbate or poloxamer surfactant; up to about 150 mM of sodiumchloride, about 1 to about 30 mM sodium phosphate; and about 0 to 5 wt %sucrose or trehalose or combination thereof; wherein the composition hasa pH of about 6.0 to about pH 7.0, and more preferably about 6.0 toabout 6.6 and most preferably about 6.3 to about 6.5.

The surfactant/sodium chloride stabilized composition is preferablycharacterized by SEC analysis at T₂ in which: monomer content is greaterthan about 80 wt. %; aggregate(s) content is less than about 3 wt %, andfragment 3 content is about 8 wt. %.

The sodium chloride-stabilized etanercept composition is preferablycharacterized by:

-   -   (a) an SEC analysis at T₄ of greater than about 90, 91, 92, 93,        94, 95, 96, or 97 wt % monomer content; and less than about 3, 2        or 1 wt % aggregate(s) content; and    -   (b) an HIC analysis at T₂ wherein the amount of the composition        represented by peak 1 of the HIC chromatogram is less than about        4, 3, 2 or 1 wt %; the amount of the composition represented by        peak 2 of the HIC chromatogram is greater than 80, 81, 82, 83,        84 85 or 86 wt %; and the amount of the composition represented        by peak 3 of the HIC chromatogram is less than about 20, 19, 18,        17, 16, 15, 14, or 13 wt %; and    -   (c) an HIC analysis at T₄ wherein the amount of the composition        represented by peak 1 of the HIC chromatogram is less than about        3, 2 or 1 wt %; the amount of the composition represented by        peak 2 of the HIC chromatogram is greater than 80, 81, 82, 83,        84 or 85 wt %; and the amount of the composition represented by        peak 3 of the HIC chromatogram is less than 20, 19, 18, 17, 16,        15, 14 or 13 wt %.

In a further embodiment, preferred composition using surfactant plussodium choride for stabilization comprise up to about 150 mM sodiumchloride, about 1 to 30 mM sodium phosphate, and about 0-5 wt. % sucroseor trehalose, or combination of sucrose and trehalose and having a pH ofabout 6.0 to 6.6; and characterized by: an SEC analysis at T₄ of greaterthan about 95 wt. % monomer content and less than about 1 wt %aggregate(s) content; an HIC analysis at T₂ wherein the amount of thecomposition represented by peak 1 of the HIC chromatogram is less thanor equal to about 3 wt %; the amount of the composition represented bypeak 2 of the HIC chromatogram is greater than about 82 wt %; and theamount of the composition represented by peak 3 of the HIC chromatogramis less than about 15 wt %; and an HIC analysis at T₄ wherein the amountof the composition represented by peak 1 of the HIC chromatogram is lessthan about 2 wt %; the amount of the composition represented by peak 2of the HIC chromatogram is greater than about 84 wt %; and the amount ofthe composition represented by peak 3 of the HIC chromatogram is lessthan or equal to about 14 wt %.

Particularly preferred compositions in terms of reduced aggregates andfragments are those in which the surfactant/sodium chloride stabilizedetanercept formulations exhibit HIC analysis at T₄ or T₂ wherein theamount of the composition represented by peak 1 of the HIC chromatogramis less than about 1%; the amount of the composition represented by peak2 of the HIC chromatogram is greater than about 95 wt. % and preferablygreater than about 99 wt %; and the amount of the compositionrepresented by peak 3 of the HIC chromatogram is less than about 1 wt %.

In a further embodiment of the invention, an NaCl stabilized etanerceptformulation contains up to about 5 mM arginine.

In the above-referenced NaCl stabilized etanercept formulations, theterms “SEC”, “T₂” “T₄” “HIC” “monomer content” “aggregate(s)” and“fragment 3” “peak 1,” “peak 2,” and “peak 3,” are defined in theexamples below.

Surfactants for Reduced Sub-Visible Particles

As noted above, the invention employs surfactants, preferablypolysorbate surfactants or poloxamer surfactants, to achieve a markedreduction in the formation of subvisible particles in an etanerceptformulation. While the invention contemplates the use of any surfactant,and any polysorbate or poloxamer surfactant, the preferred surfactantsfor use in the invention are polysorbate 80 and Pluronic F-68, both ofwhich readily commercially available. The invention may be practicedusing the surfactant additives to achieve reductions in sub-visibleparticles with or without the additional stabilizing ingredientsdescribed in the embodiments above. The present invention is based inpart on our surprising and unexpected discovery that the presencesurfactants can afford a dramatic reduction in sub-visible particles inetanercept formulations, in comparison to formulations lacking suchsurfactants.

Polysorbate surfactants contemplated for use herein are derived frompolyethoxylated sorbitan. Polysorbate 80, (also known aspolyoxyethylene-sorbitan-20 mono-oleate) is also commercially availableas “Tween 80” and is particularly preferred for use in the presentinvention. Polysorbate 20, 40 and 60 may also be used.

The poloxamer surfactants contemplated for use herein are well known inthe pharmaceutical formulation art, and consist of block copolymersbased on ethylene oxide and propylene oxide and may be commerciallyobtained from BASF. For example, BASF markets the poloxamers knowncommercially as Pluronic F-68 and which is particularly preferred foruse in the present invention.

The amount of polysorbate or poloxamer useful in the etanerceptformulations of the present invention is added to the formulations inthe range of 0.0005% to 1% (w/v); preferably within the range of about0.005% to about 0.1% (w/v), and most preferably in the range of about0.01 to about 0.05% (w/v), of the formulation.

Additional Components of the Provided Pharmaceutical Compositions

The formulations of the invention may also include buffers, tonicitymodifiers, excipients, pharmaceutically acceptable carriers and othercommonly used inactive ingredients of the pharmaceutical compositions.For simplicity, these are discussed more fully later in the application.

Buffers maintain pH in a desired range. Suitable buffers includehistidine, potassium phosphate, sodium or potassium citrate, maleicacid, ammonium acetate, tris-(hydroxymethyl)-aminomethane (tris),various forms of acetate and diethanolamine. The concentration of thebuffer in the formulation is preferably between about 1 mM to about 1 M,and more preferably about 10 mM to about 200 mM. Buffers are well knownin the art and are manufactured by known methods and available fromcommercial suppliers.

Examples of suitable buffers are phosphate, histidine, citrate, maleate,tartrate, succinate, acetate, tris-(hydroxymethyl)-aminomethane (tris),bicarbonate.

In a preferred embodiment, the buffer is sodium phosphate.

In a preferred embodiment, the pH of the pharmaceutical composition isat or near physiological levels. Thus, preferably, the pH of theprovided compositions is between about 5.8 and about 8.4; and even morepreferably, between about 6.2 and about 7.4. A person of ordinary skillin the art will understand that the pH can be adjusted as necessary tomaximize stability and solubility of etanercept in a particularformulation. Thus, etanercept formulations at a pH outside ofphysiological ranges, yet tolerable to the patient, are also within thescope of the invention.

A tonicity modifier is a molecule that contributes to the osmolality ofa solution. The osmolality of a pharmaceutical composition is preferablyadjusted to maximize the active ingredient's stability and/or tominimize discomfort to the patient upon administration. It is generallypreferred that a pharmaceutical composition be isotonic with serum,i.e., having the same or similar osmolality, which is achieved byaddition of a tonicity modifier.

In a preferred embodiment, the osmolality of the provided formulationsis from about 180 to about 420 mOsM. However, it is to be understoodthat the osmolality can be either higher or lower as specific conditionsrequire.

Examples of tonicity modifiers suitable for modifying osmolalityinclude, but are not limited to amino acids (not including arginine)(e.g., cysteine, histidine and glycine), salts (e.g., sodium chloride,potassium chloride and sodium citrate) and/or saccharides (e.g.,sucrose, glucose and mannitol).

Preferred tonicity modifiers are glycine, alanine, sodium chloride,potassium chloride, and sodium sulfate.

In a preferred embodiment, the concentration of the tonicity modifier inthe formulation is preferably between about 1 mM to about 1 M, morepreferably about 10 mM to about 200 mM. Tonicity modifiers are wellknown in the art and are manufactured by known methods and availablefrom commercial suppliers.

Excipients, also referred to as chemical additives, co-solutes, orco-solvents, that stabilize the polypeptide while in solution (also indried or frozen forms) can also be added to a pharmaceuticalcomposition. Excipients are well known in the art and are manufacturedby known methods and available from commercial suppliers.

Examples of suitable excipients include but are not limited tosugars/polyols such as: sucrose, lactose, glycerol, xylitol, sorbitol,mannitol, maltose, inositol, trehalose, glucose; polymers such as: serumalbumin (bovine serum albumin (BSA), human SA or recombinant HA),dextran, poly(viny alcohol) PVA, hydroxypropyl methylcellulose (HPMC),polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP),hydroxyethylcellulose (HEC); non-aqueous solvents such as: polyhydricalcohols, (e.g., PEG, and glycerol) and dimethylformamide (DMF); aminoacids such as: proline, L-serine, sodium glutamic acid, alanine,glycine, lysine hydrochloride, sarcosine and gamma-aminobutyric acid;and miscellaneous excipients such as: potassium phosphate, sodiumacetate, ammonium sulfate, magnesium sulfate, sodium sulfate,trimethylamine N-oxide, betaine, metal ions (e.g., zinc, calcium, andmagnesium), CHAPS, monolaurate, 2-O-beta-mannoglycerate or anycombination of the above.

Preferred excipients are sucrose, lactose, glycerol, xylitol, sorbitol,mannitol, maltose, inositol, trehalose, glucose, bovine serum albumin(BSA), human serum albumin (HSA), recombinant albumin, dextran, PVA,hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin,polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC), polyethyleneglycol, ethylene glycol, glycerol, alanine, glycine, lysinehydrochloride, sarcosine, SDS, trimethylamine N-oxide, betaine, zincions, calcium ions, magnesium ions, CHAPS, sucrose monolaurate, and2-O-beta-mannoglycerate.

The concentration of one or more excipients in a formulation of theinvention is/are preferably between about 0.001 to 5 weight percent,more preferably about 0.1 to 2 weight percent.

Methods of Treatment

In another embodiment, the invention provides a method of treating amammal comprising administering a therapeutically effective amount ofthe pharmaceutical compositions of the invention to a mammal, whereinthe mammal has a disease or disorder that can be beneficially treatedwith etanercept.

In a preferred embodiment, the etanercept is derived from the samespecies of mammal as is to be treated with the composition.

In a preferred embodiment, the mammal is a human.

Diseases or disorders that can be treated with the provided compositionsinclude but are not limited to rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis, Wegener's disease (granulomatosis),Crohn's disease (or inflammatory bowel disease), chronic obstructivepulmonary disease (COPD), Hepatitis C, endometriosis, asthma, cachexia,psoriasis, and atopic dermatitis. Additional diseases or disorders thatcan be treated with the compositions of the present invention includethose described in WO 00/62790, WO 01/62272, U.S. Patent Application No.2001/0021380, and U.S. Pat. No. 7,648,702 B2, the relevant portions ofwhich are incorporated herein by reference.

The provided pharmaceutical compositions may be administered to asubject in need of treatment by injection systemically, such as byintravenous injection; or by injection or application to the relevantsite, such as by direct injection, or direct application to the sitewhen the site is exposed in surgery; or by topical application.

In one embodiment, the invention provides a method of treatment and/orprevention of rheumatoid arthritis comprises administering to a mammalin need thereof a therapeutically effective amount of one of theprovided etanercept compositions.

The therapeutically effective amount of the etanercept in the providedcompositions will depend on the condition to be treated, the severity ofthe condition, prior therapy, and the patient's clinical history andresponse to the therapeutic agent. The proper dose can be adjustedaccording to the judgment of the attending physician such that it can beadministered to the patient one time or over a series ofadministrations.

In one embodiment, the effective etanercept amount per adult dose isfrom about 1-500 mg/m², or from about 1-200 mg/m², or from about 1-40mg/m² or about 5-25 mg/m².

Alternatively, a flat dose may be administered, whose amount may rangefrom 2-500 mg/dose, 2-100 mg/dose or from about 10-80 mg/dose.

If the dose is to be administered more than one time per week, anexemplary dose range is the same as the foregoing described dose rangesor lower and preferably administered two or more times per week at a perdose range of 25-100 mg/dose.

In another embodiment, an acceptable dose for administration byinjection contains 80-100 mg/dose, or alternatively, containing 80 mgper dose.

The dose can be administered weekly, biweekly, or separated by severalweeks (for example 2 to 8).

In one embodiment, etanercept is administered at 25 to 75 mg/ml by asingle subcutaneous (SC) injection.

In some instances, an improvement in a patient's condition will beobtained by administering a dose of up to about 100 mg of thepharmaceutical composition one to three times per week over a period ofat least three weeks. Treatment for longer periods may be necessary toinduce the desired degree of improvement. For incurable chronicconditions the regimen may be continued indefinitely. For pediatricpatients (ages 4-17), a suitable regimen may involve administering adose of 0.4 mg/kg to 5 mg/kg of etanercept, one or more times per week.

In another embodiment, the pharmaceutical formulations of the inventionmay be prepared in a bulk formulation, and as such, the components ofthe pharmaceutical composition are adjusted to be higher than would berequired for administration and diluted appropriately prior toadministration.

The pharmaceutical compositions can be administered as a soletherapeutic or in combination with additional therapies as needed. Thus,in one embodiment, the provided methods of treatment and/or preventionare used in combination with administering a therapeutically effectiveamount of another active agent. The other active agent may beadministered before, during, or after administering the pharmaceuticalcompositions of the present invention. Another active agent may beadministered either as a part of the provided compositions, oralternatively, as a separate formulation.

Administration of the provided pharmaceutical compositions can beachieved in various ways, including parenteral, oral, buccal, nasal,rectal, intraperitoneal, intradermal, transdermal, subcutaneous,intravenous, intra-arterial, intracardiac, intraventricular,intracranial, intratracheal, intrathecal administration, intramuscularinjection, intravitreous injection, and topical application.

The pharmaceutical compositions of this invention are particularlyuseful for parenteral administration, i.e., subcutaneously,intramuscularly, intravenously, intraperitoneal, intracerebrospinal,intra-articular, intrasynovial, and/or intrathecal. Parenteraladministration can be by bolus injection or continuous infusion.Pharmaceutical compositions for injection may be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. In addition, a number of recent drug deliveryapproaches have been developed and the pharmaceutical compositions ofthe present invention are suitable for administration using these newmethods, e.g., Inject-ease®, Genject®, injector pens such as GenPen®,and needleless devices such as MediJector® and BioJector®. The presentpharmaceutical composition can also be adapted for yet to be discoveredadministration methods. See also Langer, 1990, Science, 249:1527-1533.

The provided pharmaceutical compositions can also be formulated as adepot preparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the formulations may bemodified with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions may, if desired, be presented in a vial,pack or dispenser device which may contain one or more unit dosage formscontaining the active ingredient. In one embodiment the dispenser devicecan comprise a syringe having a single dose of the liquid formulationready for injection. The syringe can be accompanied by instructions foradministration.

In another embodiment, the present invention is directed to a kit orcontainer, which contains an aqueous pharmaceutical composition of theinvention. The concentration of the polypeptide in the aqueouspharmaceutical composition can vary over a wide range, but is generallywithin the range of from about 0.05 to about 20,000 micrograms permilliliter (μg/ml) of aqueous formulation. The kit can also beaccompanied by instructions for use.

The present invention is more particularly described in the followingexamples that are intended as illustrative only, since manymodifications and variations therein will be apparent to those skilledin the art. In the following examples it should be understood thatweight percentages of various ingredients are expressed as w/vpercentages.

Example 1A Etanercept Stabilized with Surfactant Plus Serine

A stable aqueous pharmaceutical composition containing etanercept andstabilized with surfactant and serine (without arginine) was prepared asfollows:

Each formulation component (buffer, amino acid, sugar, polyol,surfactant etc) is weighed to the amount required for a given volume offormulation buffer. These components are combined into a beaker orvessel capable of carrying and measuring the given volume of formulationbuffer. A volume of deionized water equal to approximately ¾ of thetarget given formulation buffer is added to the beaker, and thecomponents solubilized through use of a magnetic stir bar. The pH of thebuffer is adjusted to the target formulation pH using 1 molar sodiumhydroxide and/or 1 molar hydrogen chloride. The final formulation buffervolume is then raised to the target volume through the addition ofdeionized water. The solution is mixed with a magnetic stir bar afterfinal water addition. Etanercept protein solution is placed in dialysismaterial housing (such as Thermo Scientific Slide-A-Lyzer MINI DialysisUnit 10,000 MWCO), which is then placed in contact with the desiredformulation buffer for 12 hours at 4° C. Formulation buffer volume toprotein solution volume ratio should be no less than 1000:1. Thedialysis housing and protein solution it contains is then placed in asecond, equal volume of formulation buffer for an additional 12 hours at4° C.

Resulting protein solution is removed from the dialysis materialhousing, and the concentration of protein determined using ultravioletspectroscopy. Protein concentration is adjusted to the desired levelusing centrifugation (such as Amicon Ultra 10,000 MWCO CentrifugalConcentrators) and/or dilution with formulation buffer.

Five sample compositions of the invention in which etanercept isstabilized with serine (in the absence of arginine) are represented

Formulation 1:15

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Serine(inactive ingredient) 25 mM Sodium phosphaye, pH 6.3 (inactive) 25 mMSucrose (inactive) 1% (w/v) NaCl (inactive) 100 mM Polysorbate 80 .02%(w/v)

Formulation 1:12

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Serine(inactive ingredient) 25 mM Sodium phosphate, pH 6.4 (inactive) 25 mMSucrose (inactive) 2.5% (w/v) or 5% (w/v) NaCl (inactive) 100 mMPolysorbate 80 .02% (w/v)

Formulation 1:16

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Serine(inactive ingredient) 50 mM Sodium phosphaye, pH 6.4 (inactive) 25 mMSucrose (inactive) 5% (w/v) NaCl (inactive) 25 mM Polysorbate 80 .02%(w/v)

Formulation 2:4

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Serine(inactive ingredient) 100 mM Sodium phosphaye, pH 6.3 (inactive) 25 mMSucrose (inactive) 1% (w/v) Polysorbate 80 .02% (w/v)

Formulation 3:8

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Serine(inactive ingredient) 120 mM Sodium phosphaye, pH 6.3 (inactive) 25 mMSucrose (inactive) 1% (w/v) Polysorbate 80 .02% (w/v)The compositions can be tested for long-term stability by size exclusionchromatography (SEC), denatured SEC (dSEC), hydrophobic interactionchromatography (HIC), sodium dodecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE), and for binding and bioactivity at varioustimepoints. The bioactivity can be measured by any number of well-knownassays including by SEC, dSEC, HIC, as discussed below. Flow camanalysis can be used to evaluate sub-visible particles.

For example, the techniques of Size Exclusion Chromatography aredescribed in Hawe et al, Pharm. Res. 2011, 28: 2302 and/or vanMarrschalkerweerd et al., Eur. J. Pharm. Biopharm. 2011, 78: 213.Similarly, the techniques of Denatured Size Exclusion Chromatography,Hydrophobic Interaction Chromatography, and SodiumDodecylSulfate-PolyAcrylamide Gel Electrophoresis are also well known topersons having ordinary skill in the art.

It is believed that the composition will be stable over the term of twoyears or more.

Example 1B Etanercept Stabilized with Surfactant Plus Proline

Compositions stabilized with surfactant plus proline in this Example 1Bmay be prepared and tested using the procedures similar to thosedescribed in Example 1A. Etanercept formulations using surfactant plusproline for stabilization, exemplified below, do not contain arginine.

Formulation 1:4

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Proline(inactive ingredient) 25 mM Sodium phosphate, pH 6.3 (inactive) 25 mMSucrose (inactive) 2.5% (w/v) NaCl (inactive) 50 mM Polysorbate 80 .02%(w/v)

Formulation 1:5

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Proline(inactive ingredient) 50 mM Sodium phosphate, pH 6.3 (inactive) 25 mMSucrose (inactive) 1.0% (w/v) NaCl (inactive) 25 mM Polysorbate 80 .02%(w/v)

Formulation 1:6

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Proline(inactive ingredient) 100 mM Sodium phosphate, pH 6.3 (inactive) 25 mMSucrose (inactive) 1.0% (w/v) Polysorbate 80 .02% (w/v)

The compositions can be tested for long-term stability, and thebioactivity can be measured in the same fashion as discussed in Example1A. Flow cam analysis can be used to evaluate sub-visible particles.

It is believed that the compositions will be stable over the term of twoyears or more.

Example 1C

Etanercept Stabilized with Surfactant Plus Glutamate Compositionsstabilized with surfactant plus glutamate may be prepared and testedusing the procedures similar to those described in Example 1A.

Surfactant/Glutamate stabilized etanercept compositions, containing noarginine, are exemplified below:

Formulation 1:9

Ingredient concentration Etanercept (active ingredient) 50 mg/mlGlutamate (inactive ingredient) 25 mM Sodium phosphate, pH 6.3(inactive) 25 mM Sucrose (inactive) 1% (w/v) NaCl (inactive) 100 mMPolysorbate 80 .02% (w/v)

Formulation 2:2

Ingredient concentration Etanercept (active ingredient) 50 mg/mlGlutamate (inactive ingredient) 50 mM Sodium phosphate, pH 6.3(inactive) 25 mM Sucrose (inactive) 1% (w/v) NaCl (inactive) 50 mMPolysorbate 80 .02% (w/v)

Formulation 2:3

Ingredient concentration Etanercept (active ingredient) 50 mg/mlGlutamate (inactive ingredient) 100 mM Sodium phosphate, pH 6.3(inactive) 25 mM Sucrose (inactive) 1% (w/v) Polysorbate 80 .02% (w/v)

Formulation 3:5

Ingredient concentration Etanercept (active ingredient) 50 mg/mlGlutamate (inactive ingredient) 120 mM Sodium phosphate, pH 6.5(inactive) 25 mM Sucrose (inactive) 1% (w/v) Polysorbate 80 .02% (w/v)

The composition can be tested for long-term stability, and thebioactivity can be measured in the same fashion as discussed in Example1A. Flow cam analysis can be used to evaluate sub-visible particles.

It is believed that the composition will be stable over the term of twoyears or more.

Example 2A Etanercept Stabilized with Surfactant Plus Calcium Chloride

Etanercept formulations stabilized with surfactant plus calcium chloridemay be prepared and tested using the procedures similar to thosedescribed in Example 1A.

Etanercept compositions stabilized with surfactant plus calciumchloride, and containing no arginine, are exemplified below.

Formulation P1:1

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Calciumchloride (inactive ingredient) 2 mM Sodium phosphate, pH 6.3 (inactive)25 mM Polysorbate 80 .02% (w/v)

Formulation 1:11

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Calciumchloride (inactive ingredient) 2 mM Sodium phosphate, pH 6.3 (inactive)25 mM NaCl (inactive) 100 mM Sucrose (inert) 2.5% (w/v) Polysorbate 80.02% (w/v)

Formulation 1:18

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Calciumchloride (inactive ingredient) 2 mM Sodium phosphate, pH 6.3 (inactive)25 mM Xylitol (inactive) 10 mM Polysorbate 80 .02% (w/v)

Formulation 3:6

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Calciumchloride (inactive ingredient) 2 mM Sodium phosphate, pH 6.3 (inactive)15 mM NaCl (inactive) 75 mM Sucrose (inactive) 3% (w/v) Polysorbate 80.02% (w/v)

Formulation 3:9

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Calciumchloride (inactive ingredient) 1 mM Sodium phosphate, pH 6.6 (inactive)10 mM NaCl (inactive) 50 mM Trehalose (inactive) 5% (w/v) Polysorbate 80.02% (w/v)

The composition can be tested for long-term stability, and thebioactivity can be measured in the same fashion as discussed in Example1A. Flow cam analysis can be used to evaluate sub-visible particles.

It is believed that the composition will be stable over the term of twoyears or more.

Example 2B Etanercept Stabilized with Surfactant Plus Magnesium Chloride

Etanercept formulations stabilized with surfactant plus magnesiumchloride may be prepared and tested using the procedures similar tothose described in Example 1A. The etanercept formulations exemplifiedbelow do not contain arginine.

Formulation P1:2

Ingredient concentration Etanercept (active ingredient) 50 mg/mlMagnesium chloride (inactive ingredient) 2 mM Sodium phosphate, pH 6.3(inactive) 25 mM Polysorbate 80 .02% (w/v)

Formulation 2:15

Ingredient concentration Etanercept (active ingredient) 50 mg/mlMagnesium chloride (inactive ingredient) 4 mM Sodium phosphate, pH 6.4(inactive) 25 mM NaCl (inactive) 100 mM Sucrose (inactive) 2.5% (w/v)Polysorbate 80 .02% (w/v)

Formulation 3:7

Ingredient concentration Etanercept (active ingredient) 50 mg/mlMagnesium chloride (inactive ingredient) 5 mM Sodium phosphate, pH 6.3(inactive) 15 mM NaCl (inactive) 75 mM Sucrose (inactive) 2.5% (w/v)Polysorbate 80 .02% (w/v)

Formulation 3:14

Ingredient concentration Etanercept (active ingredient) 50 mg/mlMagnesium chloride (inactive ingredient) 10 mM Sodium phosphate, pH 6.3(inactive) 25 mM NaCl (inactive) 110 mM Sucrose (inactive) 1% (w/v)Polysorbate 80 .02% (w/v)

Formulation 4:2

Ingredient concentration Etanercept (active ingredient) 50 mg/mlMagnesium chloride (inactive ingredient) 10 mM Sodium phosphate, pH 6.5(inactive) 15 mM NaCl (inactive) 75 mM Sucrose (inactive) 3% (w/v)Polysorbate 80 .02% (w/v)

The composition can be tested for long-term stability, and thebioactivity can be measured in the same fashion as discussed in Example1A. Flow cam analysis can be used to evaluate sub-visible particles.

It is believed that the composition will be stable over the term of twoyears or more.

Example 2C Etanercept Stabilized with Surfactant Plus Zinc Chloride

Etanercept formulations stabilized with surfactant plus zinc chloridemay be prepared and tested using the procedures similar to thosedescribed in Example 1A.

The etanercept formulation exemplified below does not contain arginine.

Formulation P1:3

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Zincchloride (inactive ingredient) 2 mM Sodium phosphate, pH 6.3 (inactive)25 mM Polysorbate 80 .02% (w/v)

The composition can be tested for long-term stability, and thebioactivity can be measured in the same fashion as discussed in Example1A. Flow cam analysis can be used to evaluate sub-visible particles.

It is believed that the composition will be stable over the term of twoyears or more.

Example 3A Etanercept Stabilized with Surfactant Plus Meglumine

Etanercept compositions stabilized with surfactant plus meglumine may beprepared and tested using the procedures similar to those described inExample 1A. Surfactant/meglumine stabilized etanercept compositions,exemplified below, do not contain arginine.

Formulation 1:19

Ingredient concentration Etanercept (active ingredient) 50 mg/mlMeglumine (inactive ingredient) 5% (w/v) Sodium phosphate, pH 6.3(inactive) 25 mM Polysorbate 80 .02% (w/v)

Formulation 1:21

Ingredient concentration Etanercept (active ingredient) 50 mg/mlMeglumine (inactive ingredient) 0.49% (w/v) Sodium phosphate, pH 6.3(inactive) 25 mM Sucrose (inactive) 1% (w/v) NaCl (inactive) 100 mMPolysorbate 80 .02% (w/v)

The composition can be tested for long-term stability, and thebioactivity can be measured in the same fashion as discussed in Example1A. Flow cam analysis can be used to evaluate sub-visible particles.

It is believed that the composition will be stable over the term of twoyears or more.

Example 3B

Etanercept Stabilized with a Surfactant plus Derivative of MannitolEtanercept compositions stabilized with a derivative of mannitol may beprepared and tested using the procedures similar to those described inExample 1A.

The formulation exemplified below does not contain arginine:

Ingredient % by weight Etanercept (active ingredient) 50 mg/mlMannosylglycerate (inactive ingredient) 4% (w/v) Polysorbate 80 .02%(w/v)

The composition can be tested for long-term stability, and thebioactivity can be measured in the same fashion as discussed in Example1A. Flow cam analysis can be used to evaluate sub-visible particles.

It is believed that the composition will be stable over the term of twoyears or more.

Example 4A Etanercept Stabilized with Surfactant Plus Trehalose (orSucrose) and Mannitol

Etanercept compositions stabilized with mannitol in combination withtrehalose (or sucrose), may be prepared and tested using the proceduressimilar to those described in Example 1A. The stabilized formulationsexemplified below do not contain arginine.

Formulation P1:5

Ingredient concentration Etanercept (active ingredient) 50 mg/mlTrehalose (inactive ingredient) 4% (w/v/) Mannitol (inactive) 2% (w/v/)Polysorbate 80 .02% (w/v)

Formulation 1:10

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sodiumphosphate, pH 6.32 (inactive) 25 mM Sucrose (inactive) 5% (w/v) Mannitol(inactive) 2% (w/v/) Polysorbate 80 .02% (w/v)The composition can be tested for long-term stability, and thebioactivity can be measured in the same fashion as discussed in Example1A. Flow cam analysis can be used to evaluate sub-visible particles.

It is believed that the composition will be stable over the term of twoyears or more.

Example 4B Etanercept Stabilized with Surfactant Plus Sucrose andSorbitol

Etanercept compositions stabilized with a combination of surfactant plussucrose and sorbitol may be prepared and tested using the proceduressimilar to those described in Example 1A. The formulation exemplifiedbelow does not contain arginine.

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sucrose(inactive ingredient) 4% (w/v) Sorbitol (inactive) 2% (w/v) Sodiumphosphate, pH 6.3 (inactive) 25 mM Polysorbate 80 .02% (w/v)

The composition can be tested for long-term stability, and thebioactivity can be measured in the same fashion as discussed in Example1A. Flow cam analysis can be used to evaluate sub-visible particles.

It is believed that the composition will be stable over the term of twoyears or more.

The foregoing description of the exemplary embodiments of the inventionhas been presented only for the purposes of illustration and descriptionand is not intended to be exhaustive or to limit the invention to theprecise forms disclosed. Many modifications and variations are possiblein light of the above teaching.

Example 5 Etanercept Stabilized with Surfactant Plus Xylitol

Etanercept formulations stabilized with surfactant plus xylitol may beprepared and tested using the procedures similar to those described inExample 1A. The compositions exemplified below do not contain arginine.

Formulation 1:17

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sodiumphosphate, pH 6.3 (inactive) 25 mM Xylitol (inactive) 10% (w/v)Polysorbate 80 .02% (w/v)

Formulation 2:10

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sodiumphosphate, pH 6.31 (inactive) 25 mM Xylitol (inactive) 6% (w/v)Polysorbate 80 .02% (w/v)

Formulation 2:11

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sodiumphosphate, pH 6.3 (inactive) 25 mM Xylitol (inactive) 2.5% (w/v) Sucrose(inactive ingredient) 5% (w/v) Polysorbate 80 .02% (w/v)

Formulation 2:18

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sodiumphosphate, pH 6.4 (inactive) 25 mM Xylitol (inactive) 2.5% (w/v)Meglumine (inactive) 2.5% (w/v) Polysorbate 80 .02% (w/v)

Formulation 2:19

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sodiumphosphate, pH 6.24 (inactive) 10 mM Xylitol (inactive) 2.5% (w/v)Meglumine (inactive) 2.5% (w/v) NaCl (inactive) 2.5% (w/v) Sucrose(inactive) 1% (w/v) Polysorbate 80 .02% (w/v)

Example 6 Etanercept Stabilized with Surfactant and NaCl

Etanercept formulations stabilized with surfactant and NaCl, alone, orNaCl in combination with sucrose, trehalose and/or arginine, may beprepared and tested using the procedures similar to those described inExample 1A. With the exception of formulation 3:13 below, thecompositions exemplified below do not contain arginine.

Formulation 2:8

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sodiumphosphate, pH 6.32 (inactive) 25 mM NaCl (inactive) 150 mM Polysorbate80 .02% (w/v)

Formulation 2:6

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sodiumphosphate, pH 6.3 (inactive) 15 mM NaCl (inactive) 100 mM Sucrose(inactive) 2% (w/v) Polysorbate 80 .02% (w/v)

Formulation 3:10

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sodiumphosphate, pH 6.57 (inactive) 10 mM NaCl (inactive) 75 mM Sucrose(inactive) 3% (w/v) Polysorbate 80 .02% (w/v)

Formulation 3:11

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sodiumphosphate, pH 6.30 (inactive) 25 mM NaCl (inactive) 75 mM Trehalose(inactive) 3% (w/v) Polysorbate 80 .02% (w/v)

Formulation 3:12

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sodiumphosphate, pH 6.3 (inactive) 25 mM NaCl (inactive) 75 mM Sucrose(inactive) 3% (w/v) Polysorbate 80 .02% (w/v)

Formulation 3:13

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sodiumphosphate, pH 6.3 (inactive) 25 mM NaCl (inactive) 120 mM Sucrose(inactive) 1% (w/v) Arginine (inactive) 5 mM Polysorbate 80 .02% (w/v)The compositions can be tested for long-term stability, and thebioactivity can be measured in the same fashion as discussed in Example1A. Flow cam analysis can be used to evaluate sub-visible particles.

Analysis of Etanercept Formulations

A. Thermal Stability Storage

Following dialysis and concentration, samples of the etanerceptformulations exemplified above were sterile filtered in a bio safetycabinet. Using sterilized pipettes and autoclaved pipette tips, samplesof the etanercept formulations were transferred to pre-labeled andautoclaved 1 mL lyophilization vials. Vials were stoppered with sterilebutyl stoppers and crimped with aluminum caps. All vials were thentransferred to thermal stability ovens. Samples were subject to twothermal stability regimes: (1) two weeks at 40° C. and (2) four weeks at25° C. Throughout this specification, these two temperature regimes aredenoted “T₂” and T₄,” respectively.

B. Size Exclusion Chromatography (SEC)

Etanercept formulations disclosed herein were analyzed using the wellknown technique of Size Exclusion Chromatography (SEC), ahigh-performance liquid chromatography method in which analytes areseparated by size (see Rogner, M. (2000). Size Exclusion Chromatography.Protein Liquid Chromatography. M. Kastner. Amsterdam, Elsevier. 61:89-145.). In order to evaluate thermal stability of the Etanerceptsamples described above, the samples were examined by a SEC method basedon the literature (van Maarschalkerweerd, A., G. J. Wolbink, et al.(2011). “Comparison of analytical methods to detect instability ofetanercept during thermal stress testing.” European Journal ofPharmaceutics and Biopharmaceutics 78(2): 213-221.) The mobile phasebuffer was prepared to contain 50 mM sodium phosphate monobasicmonohydrate and 150 mM arginine. The pH was adjusted to 6.5 using 1 MHCl. All separations were performed using a Tosoh TSK-Gel SWxl 6 mm×4 cmguard column (cat. no. 8543) attached linearly to a Tosoh TSK-Gel G4000SWxl 7.8 mm×30 cm (cat. no. 8542). To perform a separation, the columnswere brought to room temperature (23° C.) and equilibrated with mobilephase at a flow rate of 0.5 mL/min. 5 microliters of 50 mg/mL etanerceptformulation were injected onto the column using an autosampler. Theseparation was accomplished over 30 minutes at a flow rate of 0.5mL/minute. Column eluent was monitored at a wavelength of 280 nm duringthis time.

C. Integration of Size Exclusion Chromatography Chromatograms

All integration was performed using Chromeleon software (Dionex). Priorto integration, the SEC chromatogram for a buffer containing noetanercept was subtracted from all chromatograms. All integration wasperformed between retention times of 12 minutes and 26 minutes. Severalparameters were used to define a peak. The minimum area for a detectedpeak was set to 0.05 mAu*min. The two-dimensional sensitivity for peakdetection was set to 0.01 mAu and 75 seconds. Peak shoulders were addedmanually using a manual integration tool. All detected peaks weremanually adjusted in two steps. First, peak baselines (the bottomboundary of the peak) were adjusted to horizontal. Secondly, thevertical positions of the peak baselines were adjusted to that of thechromatogram baseline. The chromatogram baseline value was defined asthe signal in absence of analyte. The signal in absence of analyte wasdefined as the absorbance in mAu at 12 minutes retention time.

D. SEC Fractions of Etanercept Formulations

In the SEC analysis of etanercept formulations described above, threeSEC chromatography fractions were identified and studied. The fractionsthat were analyzed were, in the order of elution from the SEC column:(1) a very high molecular weight fraction representing aggregates of theintact etanercept TNFR:FC fusion protein likely assembled vianon-covalent electrostatic attraction among intact etanercept molecules(hereinafter “aggregate(s)” or aggregate(s) content); (2) monomercontent, representing the intact etanercept TNFR:FC fusion protein(hereinafter referred to as “monomer” of “monomer content”); (3) afraction likely representing one fragment or a population of fragmentsof the etanercept molecule in which one portion of the TNFR:moleculefusion protein has become disassociated from the monomer; such as forexample dissociation of an arm of the FC portion of the fusion proteinat the hinge region of the molecule (hereinafter referred to as“Fragment 3”). The following table shows the relative amounts ofAggregates, Monomer and Fragment 3 determined by SEC analysis asdescribed above.

E. FlowCAM® Analysis of Sub-Visible Particles.

Method development may be done using a Manuel Prime with Non-Sampleprocedure (liquid to liquid interphase). Pronounced mixing effects areseen in the flow cell so an alternative air gap procedure (Manual Primewith Sample) is selected for sample evaluation.

Formulations of Examples 3.5 and 4.2 were evaluated with and without PS80 at “T0”. The term “T0” as used with respect to the flowCAM analysisdenotes that samples were not subjected to any appreciable thermal orstorage stress (i.e, maintained at 5C or less) and then promptly testedusing the flowCAM analysis within 24 to 72 hours) after the testedformulations were prepared.

Instrumentation & Accessories

-   FlowCAM Instrument: Model VS1, Serial #551 with Sony SX90 camera and    C70 pump with a 1 mL syringe (Fluid Imaging Technologies)-   FlowCAM Software: DSP Firmware Version: 54; version 3.0.3-   Flow Cell: Field of View (FOV FC80) with a depth of 80 μm and a    width of 700 μm (Fluid Imaging Technologies)-   Objective: 10×

Context Setting (Method & Setting Parameters)

Method: Manual Prime with Sample (air gap)Sample Analysis: 0.200 mL volume with 0.170 mL analyzedFlow Rate: 0.100 ml/minAuto image rate: 22 frames per second

Efficiency: 38.7%

Run time: 1.7 minutes

Distance to Nearest

Neighbor: 0 micronsClose Hole: 5 iterationsImages: Collage image border padding of 5

Particle Segmentation: Dark Threshold 15.00, Light Threshold 15.00Acceptable Region: Left 15, Right 1255, Top 0, Bottom 959 Camera:Shutter 8 Gain 57

Auto image rate: 22 frames per secondFlash camera delay: 100 microsecondsFlash duration: 18.5 microsecondsDiameter (ESD): Min 2.00, Max 1000.00 microns

Prior to running samples, the flow cell and objective are installed andthe Field of View and Focus optimization are performed. Systemqualification includes running water blanks and particle size standardin multiple replicates. Prior to running samples a cleaning procedure isundertaken to ensure particle counts are at acceptable levels typicallybelow 1000 particles/mL between samples or less than 5% of the sampleparticles/mL between replicate samples. The routine cleaning processuses water (Millipore Direct-Q type 1, 0.22 μm filtered, 18.2 MΩ)between cleaning agents and as a final flush prior to determining countlevels. Once the particle count reaches an acceptable particle per mLlevel the sample is carefully pipetted into the sample tip and loadedinto the flow prior to initiating the sample analysis. Run quality isdetermined during and immediately after each run using a series ofdiagnostic tools in VisualSpreadSheet including x-y capture plot (tovisualize flow pattern dynamics), aspectic ratio to diameter size plot(identify stuck particles), image review during the run and imageanalysis at completion of run using various particle characteristics(e.g. size, circularity, length, aspect ratio). Individual particle sizeis determined with Fluid Imaging Technologies software measurementtechnique known as Equivalent Spherical Diameter (ESD). ESD is the meanferet measurement of the particle based on 36 sample measurements(conducted every) 5°. A feret measurement is the perpendicular distancebetween parallel tangents touching opposite sides of the particle.

TABLE 1 SEC ANALYSIS OF MONOMER Formulation No. t₀ t₁ t₂ CommercialEnbrel ® 98.81 92.58 87.64 (comparative) [1:2] 1:5 98.38 91.65 86.89 1:998.48 92.05 86.06 1:10 98.25 91.84 84.51 1:11 98.60 92.08 89.71 1:1798.02 93.90 87.53 1:18 98.27 92.89 88.21 1:19 98.10 91.94 86.06 1:2198.22 90.78 85.43 2:2 98.11 — 86.92 2:3 98.14 — 88.84 2:4 98.12 — 88.162:6 98.09 — 87.77 2:8 98.07 — 88.38 2:10 98.09 — 87.56 2:11 98.10 —88.03 2:15 98.18 — 88.22 2:18 98.10 — 89.19 2:19 98.19 — 89.63 3:5 98.35— 90.75 3:6 98.07 — 90.75 3:7 98.09 — 89.60 3:8 98.15 — 89.27 3:9 97.90— 91.44 3:10 98.16 — 89.77 3:11 98.32 — 89.87 3:12 98.33 — 90.92 3:1398.18 — 90.74 3:14 98.22 — 90.54 4:2 98.62 90.47 Note: Amounts reportedTables I, II and III are percentages by weight T₀ = formulationmaintained at 5 C. and analyzed within 24 hours of creation. T₁ =formulation stored for one week at 40° C. T₂ = formulation stored fortwo weeks at 40 C.

TABLE II SEC ANALYSIS OF AGGREGATES Formulation No. t₀ t₁ t₂ CommercialEnbrel ® 0.09 0.59 1.02 (comparative) 1:5 0.23 0.63 1.01 1:9 0.18 0.672.20 1:10 0.26 0.68 0.82 1:11 0.12 0.50 0.64 1:17 0.31 0.70 2.17 1:180.24 0.65 1.61 1:19 0.26 0.63 1.50 1:21 0.23 0.64 1.30 2:2 0.29 — 3.532:3 0.29 — 2.31 2:4 0.29 — 2.29 2:6 0.30 — 1.81 2:8 0.30 — 1.42 2:100.29 — 2.57 2:11 0.31 — 1.68 2:15 0.27 — 1.83 2:18 0.29 — 1.53 2:19 0.26— 1.24 3:5 0.28 — 0.99 3:6 0.23 — 1.27 3:7 0.28 — 0.93 3:8 0.28 — 1.603:9 0.37 — 0.73 3:10 0.27 — 1.33 3:11 0.20 — 1.24 3:12 0.21 — 0.85 3:130.28 — 0.86 3:14 0.25 — 0.91 4:2 1.56

TABLE III ANALYSIS OF FRAGMENT 3 Formulation No t₀ t₁ t₂ CommercialEnbrel ® 0.00 3.30 6.29 (comparative) [1:2} 1:5 0.00 4.43 6.64 1:9 0.003.96 6.34 1:10 0.00 3.78 8.04 1:11 0.00 3.92 4.71 1:17 0.00 2.33 4.101:18 0.00 3.05 4.65 1:19 0.00 3.82 6.73 1:21 0.00 4.92 7.37 2:2 0.00 —4.67 2:3 0.00 — 3.61 2:4 0.00 — 3.61 2:6 0.00 — 4.73 2:8 0.00 — 6.292:10 0.00 — 5.10 2:11 0.00 — 5.68 2:15 0.00 5.56 2:18 0.00 4.24 2:190.00 4.34 3:5 0 3.15 3:6 0 4.72 3:7 0 4.37 3:8 0 3.61 3:9 0 3.48 3:10 03.76 3:11 0 3.59 3:12 0 3.68 3:13 0 3.88 3:14 0 3.83 4:2 5.40

TABLE IV SEC MONOMER CONTENT (4 weeks/25° C.) T₀ Monomer T₄ MonomerFORMULATION No. Content Content Commercial Enbrel ® 98.15 97.86(comparative) 3:5 98.35 95.16 3:6 98.07 94.84 3:7 98.09 97.75 3:8 98.1597.65 3:9 97.90 97.44 3:10 98.16 97.66 3:11 98.32 97.75 3:12 98.33 97.903:13 98.18 97.78 3:14 98.22 97.79 4:2 98.62 94.70Table IV below shows monomer (etanercept) content of etanerceptformulations prepared according to the present invention, when storedfor four weeks at 25 C.°-denoted by the symbol T₄. In the followingtable T₀ represents SEC measurements conducted within 24 hours offormulation preparation, at sample temperature of 5° C.; and T₄represents etanercept formulation samples subjected to SEC analysisafter 4 weeks storage at 25° C.

TABLE V SEC AGGREGATES CONTENT (4 weeks/25° C.) T₀ Aggregate(s) T₄Aggregate(s) FORMULATION No. Content Content Commercial Enbrel ® 0.280.25 (comparative) 3:5 — 0.50 3:6 — 0.57 3:7 0.28 0.31 3:8 0.28 0.37 3:90.37 0.41 3:10 0.27 0.32 3:11 0.20 0.27 3:12 0.21 0.26 3:13 0.28 0.323:14 0.25 0.28 4:2 — 0.57

Table V below shows aggregate(s) content of etanercept formulationsprepared according to the present invention after storage for four weeksat 25 C.°. In the following table T₀ represents SEC measurementsconducted within 24 hours of formulation preparation, at sampletemperature of 5° C.; and T₄ represents etanercept formulation samplessubjected to SEC analysis after 4 weeks storage at 25° C.

HIC Analysis of Etanercept Formulations

The following tables (Tables VI and VII) show the results of hydrophobicinteraction chromatography (“HIC chromatography”) conducted on samples3:5 through 3:14. HIC chromatography was carried out in the mannerdescribed in U.S. Pat. No. 7,294,481, incorporated herein by reference.Samples were evaluated at t₀ (within 24 hours of preparation at 5° C.)and again after either two weeks of storage at 25° C. (t₂) (see TableVI) or after 4 weeks of storage at 25° C. (t₄)(See Table VII) Peak 1 isbelieved to be or include “Fragment 3” referenced above in thediscussion of SEC data; Peak 2 is etanercept monomer as referenced abovein the discussion of SEC data; and Peak 3 represents or includes“Aggregate(s)” as referenced above in the discussion of SEC data. Itshould further be understood that the terms “peak 1”, “peak 2” and “peak3 as used here also constitute a reference to the HIC peak 1, peak 2 andpeaks referred to and disclosed in FIG. 4 of U.S. Pat. No. 7,294,481incorporated herein by reference.

TABLE VI HIC Data after Two Weeks Storage at 40° C. PEAK 1 PEAK 2 PEAK 3Form. # T₀ T₂ T₀ T₂ T₀ T₂ Commercial Enbrel ® 0.91 3.23 86.72 83.4112.33 13.36 (comparative) 3:5 0.72 2.95 85.82 82.50 13.45 14.55 3:6 0.723.44 85.91 83.26 13.36 13.30 3:7 0.74 3.52 86.11 82.41 13.15 14.07 3:80.72 3.08 85.80 83.90 13.48 13.02 3:9 0.69 2.39 90.93 85.09 8.38 12.523:10 0.74 3.06 87.36 84.24 11.90 12.70 3:11 0.56 3.10 86.46 83.73 12.9813.18 3:12 0.68 3.07 86.80 83.52 12.52 13.40 3:13 0.77 2.86 86.45 84.3312.78 12.82 3:14 0.71 2.51 87.14 84.54 12.15 12.95

TABLE VII HIC Data after Storage at 25° C. for 4 Weeks PEAK 1 PEAK 2PEAK 3 Form. # T₀ T₄ T₀ T₄ T₀ T₄ Commercial Enbrel ® 0.91 1.09 86.7686.95 12.33 11.97 (comparative) 3:5 0.54 1.10 85.12 84.06 14.33 14.843:6 0.55 1.40 85.50 84.07 13.96 14.53 3:7 0.74 1.63 86.11 85.65 13.1512.72 3:8 0.72 1.20 85.80 85.98 13.48 12.82 3:9 0.69 1.05 90.93 86.468.38 12.50 3:10 0.74 1.03 87.36 85.83 11.90 13.14 3:11 0.56 1.11 86.4685.32 12.98 13.57 3:12 0.68 0.81 86.80 86.36 12.52 12.83 3:13 0.77 1.0186.45 85.78 12.78 13.21 3:14 0.71 1.13 87.14 85.58 12.15 13.29 4:2 0.631.38 85.16 84.38 14.21 14.25

TABLE VIII FLOW CAM ANALYSIS OF SUBVISIBLE PARTICLES (number ofparticles per ml) Particle size Particle size Particle size Particlesize Particle size Particle size Particle size 1-2 μm 2-5 μm 5-10 μm10-15 μm 15-25 μm 25-40 μm 40-50 μm Form 4.2 1000/ml ± 230     5600/ml ±250    2000/ml ± 490    300/ml ± 71    91/ml ± 64    24/ml ± 9     7/ml± 13  (without PS80) Form 3.5 2100 ± 1100  8000 ± 3900 2300 ± 940  240 ±210 120 ± 84  60 ± 66 27 ± 34 (without PS 80) Enbrel ® 270 ± 150 1400 ±670  500 ± 190 120 ± 50 76 ± 41 27 ± 10 0 ± 0 Form 4.2 230 ± 130 1200 ±440  470 ± 310  98 ± 87 49 ± 57 16 ± 17 5 ± 9 (with PS80 Form 3.5 540 ±120 2600 ± 440 1000 ± 290 240 ± 81 44 ± 34 11 ± 9  5 ± 9 (with PS80)Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. A method of treating a subject suffering fromrheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis,granulomatosis, Crohn's disease, inflammatory bowel disease, chronicobstructive pulmonary disease (COPD), Hepatitis C, endometriosis,asthma, cachexia, psoriasis, or atopic dermatitis, comprisingadministering to the subject an aqueous pharmaceutical compositioncomprising: etanercept at 25 to 75 mg/ml; a surfactant at 0.01 to 0.05%w/v; a metal ion selected from the group consisting of calcium,magnesium and zinc, wherein the metal ion is present at 2 mM to 20 mM; abuffer selected from the group consisting of phosphate, histidine,citrate, maleate, tartrate, succinate,tris-(hydroxymethyl)-aminomethane, and bicarbonate; wherein the aqueouspharmaceutical composition has a pH of between 6 and 7, and the aqueouspharmaceutical composition is free of arginine.
 2. The method of claim1, wherein the surfactant is polysorbate 80, polysorbate 60, polysorbate40, or polysorbate
 20. 3. The method of claim 1, wherein the surfactantcomprises polysorbate
 80. 4. The method of claim 1, wherein the aqueouspharmaceutical composition has (i) less than 2500 particles/ml having asize of 5-10 μm, (ii) less than 3500 particles having size of about 2-5μm, and (iii) less than 700 particles/ml having size of about 1-2 μm. 5.The method of claim 1, wherein etanercept is present at 25 to 50 mg/ml,the metal ion is magnesium, and wherein the aqueous pharmaceuticalcomposition further comprises sucrose or trehalose at 0.5 to 6 wt %,NaCl at 25 to 150 mM, and the buffer is sodium phosphate at 1 to 30 mM.6. The method of claim 1, wherein the metal ion is magnesium and whereinthe aqueous pharmaceutical composition further comprises NaCl at 0 to100 mM; and the buffer is sodium phosphate at 1 to 30 mM.
 7. The methodof claim 1, wherein the aqueous pharmaceutical composition ischaracterized by: (a) an SEC analysis at T₄ of greater than 90 wt %monomer content; and less than 3 wt % aggregate(s) content; and (b) anHIC analysis at T₂ wherein the amount of the aqueous pharmaceuticalcomposition represented by peak 1 of the HIC chromatogram is less than 4wt %; the amount of the aqueous pharmaceutical composition representedby peak 2 of the HIC chromatogram is greater than 80 wt %; and theamount of the aqueous pharmaceutical composition represented by peak 3of the HIC chromatogram is less than 20 wt %; and (c) an HIC analysis atT₄ wherein the amount of the aqueous pharmaceutical compositionrepresented by peak 1 of the HIC chromatogram is less than 3 wt %; theamount of the aqueous pharmaceutical composition represented by peak 2of the HIC chromatogram is greater than 80, wt %; and the amount of theaqueous pharmaceutical composition represented by peak 3 of the HICchromatogram is less than 20 wt %.
 8. The method of claim 1, wherein theaqueous pharmaceutical composition is administered subcutaneously. 9.The method of claim 1, wherein the aqueous pharmaceutical composition isadministered intramuscularly or intravenously.
 10. The method of claim1, wherein the aqueous pharmaceutical composition is administered onceweekly.
 11. The method of claim 1, wherein the aqueous pharmaceuticalcomposition is administered twice weekly.
 12. The method of claim 1,wherein the aqueous pharmaceutical composition has about 1 wt %aggregate content or less than 1 wt % aggregate content as determined bysize exclusion chromatography after 4 weeks storage at 25° C.
 13. Themethod of claim 1, comprising administering 25-100 mg etanercept in theaqueous pharmaceutical composition.
 14. A method of treating a subjectsuffering from rheumatoid arthritis, psoriatic arthritis, ankylosingspondylitis, granulomatosis, Crohn's disease, inflammatory boweldisease, chronic obstructive pulmonary disease (COPD), Hepatitis C,endometriosis, asthma, cachexia, psoriasis, or atopic dermatitis,comprising administering to the subject an aqueous pharmaceuticalcomposition comprising: etanercept at or about 50 mg/ml; up to 0.05% w/vsurfactant; 2 mM to 20 mM metal ion; about 25 mM to about 100 mM NaCl;10 mM to 200 mM of a phosphate or citrate buffer; wherein the aqueouspharmaceutical composition has a pH of 6 to 7, wherein the aqueouspharmaceutical composition is free of arginine, wherein said aqueouspharmaceutical composition has about 1 wt % aggregate content or lessthan 1 wt % aggregate content as determined by size exclusionchromatography after 4 weeks storage at 25° C., and wherein the aqueouspharmaceutical composition has: (i) less than 2500 particles/ml having asize of about 5-10 μm; (ii) less than 3500 particles having size ofabout 2-5 μm, and (iii) less than 700 particles/ml having size of about1-2 μm.
 15. The method of claim 14, wherein the aqueous pharmaceuticalcomposition further comprises sucrose or trehalose at 0.5 to 6 wt %. 16.The method of claim 14, wherein the aqueous pharmaceutical compositionis administered subcutaneously.
 17. The method of claim 14, wherein theaqueous pharmaceutical composition is administered intramuscularly orintravenously.
 18. The method of claim 14, wherein the aqueouspharmaceutical composition is administered once weekly.
 19. The methodof claim 14, wherein the aqueous pharmaceutical composition isadministered twice weekly.
 20. The method of claim 14, comprisingadministering 25-100 mg etanercept in the aqueous pharmaceuticalcomposition.